Diagnosing genetic disorders

ABSTRACT

This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as nucleic acids from biological samples, cells from bodily fluids, and nascent proteins from translation reactions. Agents comprise a detectable moiety bound to a photoreactive moiety. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals. Useful compositions include biological agents such as nucleic acids, proteins, lipids and cytokines. Conjugates can also be used to monitor the pathway and half-life of pharmaceutical composition in vivo and for diagnostic, therapeutic and prophylactic purposes. The invention also relates to kits comprised of agents and conjugates that can be used for the detection of diseases, disorders and nearly any individual substance in a complex background of substances.

REFERENCE TO RELATED APPLICATIONS

This is a Continuation of application(s) 09/504,001 filed on Feb. 14,2000 now U.S. Pat. No. 6,589,736 which is a continuation of applicationSer. No. 08/479,389 filed on Jun. 7, 1995 which is now issued U.S. Pat.No. 6,057,096 dated on May 20, 2000, which is a continuation ofapplication Ser. No. 08/345,807 filed on Nov. 22, 1994 which is nowissued U.S. Pat. No. 5,986,076, which is a continuation-in-part ofapplication Ser. No. 08/240,511 filed on May 11, 1994 which is nowissued U.S. Pat. No. 5,643,722 dated Jul. 1, 1997.

RIGHTS IN THE INVENTION

This invention was made with United States Government support undergrant number EM4727-03, awarded by the National Institutes of Health,and grant number DAAL03-92-G-0172, awarded by the Army Research Office,and the United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to agents and conjugates used in the detectionand isolation of targets from heterologous mixtures. Agents comprise adetectable moiety bound to a photoreactive moiety. Conjugates compriseagents which are coupled to substrates by one or more covalent bonds.These bonds can be easily and selectively cleaved or photocleaved withthe application of electromagnetic radiation. Substrates which may becoupled to agents include amino acids, peptides, proteins, nucleotides,nucleic acid primers for PCR reactions and lipids. The invention alsorelates to rapid and efficient methods for the detection and isolationof targets, such as cells, nucleic acids and proteins, and to kits whichcontain these components.

2. Description of the Background

Basic scientific techniques including some of the major breakthroughs inmolecular biology, chemistry and medicine have certain features incommon. Two of these features are the specific detection and isolationof individual components from complex mixtures. For example,electrophoresis and chromatography are each widely utilized proceduresto detect or isolate macromolecules from biological samples. Theseprocedures take advantage of unique or identifiable molecular propertiesof the components to be isolated such as charge, hydrophobicity andmolecular weight, to characterize and identify macromolecules. Dependingon their method of isolation, macromolecules isolated can often beutilized as products in downstream processes.

Some of the more useful detection and isolation procedures takeadvantage of physical properties of the element of interest, thesubstrate, or of molecules which can be easily attached to substrates.One of the most widely used of these properties is radioactivity andradioactive labeling with radionuclides. For the most part, substancesare not naturally radioactive and can be labeled with radioactive atoms,referred to as radionuclides, and detected using standard and well-knownradiographic procedures. Radioactive elements are detectable becausethey emit large amounts of energy in the form of alpha, beta or gammarays as they decay. Radioactivity is generally useful for labelingbecause the label is not affected by the physical state or chemicalcombination of the substance to be labeled. In addition, the specificradiation emitted can be identified by the nature of the radiation (e.g.α, β or γ), its energy and the half-life of the process. Targets can beidentified in complex mixtures from the radiation profile emitted.Further, radioactively labeled substances can be followedradiographically in chemical pathways and biological systems.

Unfortunately, radioactivity is a hazard to both human health and theenvironment. The protection which must be afforded each worker issubstantial. Special laboratory procedures, dedicated facilities andequipment, detailed record keeping and special training of laboratorypersonnel are all required for the safe use of radionuclides. Productionof radioactive reagents is also very expensive as is safe disposal whichdrives up the cost of all experiments involving radioactive agents.Further, under present guidelines, all users of radioactivity requirespecialized supervision and federal regulations must be strictly andcarefully adhered to requiring an enormous amount of record keeping.

Radioactive labeling methods also do not always provide a means ofisolating products in a form which can be further utilized. The presenceof radioactivity compromises utility for further biochemical orbiophysical procedures in the laboratory and in animals. This is clearin the case of in vitro or in vivo expression of proteinsbiosynthetically labeled with radioactive amino acids or tagged withother radioactive markers. The harm or at least potential harm of theradioactivity outweighs the benefits which might be produced by theprotein composition.

Disposal of radioactive waste is also of increasing concern both becauseof the potential risk to the public and the lack of radioactive wastedisposal sites. In addition, the use of radioactive labeling is timeconsuming, in some cases requiring as much as several days for detectionof the radioactive label. The long time needed for such experiments is akey consideration and can seriously impede research productivity. Whilefaster methods of radioactive detection are available, they areexpensive and often require complex image enhancement devices.

There are many other detectable physical properties which can exist inchemicals and chemical moieties that can be used to detect and isolatesubstances. One of these physical properties is the property ofluminescence which includes the phenomena of fluorescence andphosphorescence. Fluorescent chemicals emit radiation due to the decayof the molecule which has been excited to a higher electronic state dueto the absorption of radiation. Phosphorescent molecules can emitradiation for a much longer time intervals. Detection of the specificwavelength of radiant energy emitted allows for the detection of targetswhich may be associated with the luminescent chemicals.

Bioluminescence is rapidly becoming a widely used method for labelingmany different types of compounds. Basically, a reduced substrate isreacted with oxygen and converted into an oxidized product with anelevated or excited electronic state. The excited molecule decays to theground state and in the process, emits photons of light. This processhas been found to occur in several strains of bacteria and fungi, inmarine invertebrates such as sponges, and in shrimp and jellyfish.Bacteria which emit light are often found living symbiotically with fishin special luminescent organs. A wide variety of terrestrial organismssuch as earthworms, centipedes and insects also possess bioluminescentproperties.

One group of compounds which undergo oxidation with the emission oflight are referred to as luciferins although their individual structuremay vary. The oxidized products are termed oxy-luciferins and theenzymes which catalyze the process luciferases. The overall process isendothermic requiring chemical energy stored in one to two molecules ofadenosine triphosphate (ATP) per photon of light produced. Two types ofluciferase systems that have been widely used in molecular biology arethe bacterial system (Vibrio harveyi or V. fischeri) and the fireflysystem (Photinus pyralis).

Other labels which impart detectable properties to a substrate includechemicals with a unique absorption spectrum, electron spin resonancespectrum, optical activity, Raman spectrum or resonance Raman. spectrumSuch labels are widely used in many fields including medicine, molecularbiology and chemistry. For example, the visible or infrared absorptionspectrum of a molecule often constitutes a unique fingerprint whichallows the molecule to be identified even in the presence of a complexmixture. In the case of visible absorption, the molecule absorbs radiantenergy over a specific wavelength range because of the presence of anexcited electronic state of the molecule whose energy of transition fromthe ground state falls in the range 1.5-3 eV. In the case of infraredabsorption, bands are detected due to the excitation of vibrationalmodes of the molecule. The frequency of these bands provides informationabout the presence or absence of characteristic molecular groups such asdisulfides, carbonyls and aromatic groups.

In another application of the spectroscopic properties of molecules,nuclear magnetic resonance (NMR) spectroscopy has been extensively usedto identify specific molecules in a mixture. The nuclei of atoms, suchas protons in hydrogen atoms, which possess a net magnetic moment, willalign when placed into a magnetic field with that field and will precessabout that field with a frequency (the Lamar frequency) dependent on theindividual properties of the particle. To determine the NMR spectrum, asample of protons is placed within a strong magnetic field andirradiated with a range of radio frequencies at a 90° angle with respectto the main field. This treatment causes all the protons in the sampleto absorb energy at their characteristic frequency, flipping theirmagnetic orientations 90° with respect to their original state. Afterthe applied field is switched off, the molecules gradually relax toprecess about the main field. Receiver coils which surround the sampledetect the frequencies of precessing spins as a set of oscillatingelectric currents which constitute the NMR signal.

All of these methods suffer from similar disadvantages. For the mostpart, targets do not have unique detectable properties such as inherentradioactivity or fluorescence. Labels must be attached which arethemselves detectable and therefore make the target detectable. However,the labeling process can result in a labeled product that is in some waypermanently damaged. For example, fluorescent chemicals can be extremelytoxic to cells. Long term exposure can result in a high degree of celldeath. Often, the labeling compound may have detrimental effects on atarget's structure or activity. Protein structure is often adverselyaffected by the attachment of a detectable chemical moiety. Labeling ofnucleic acids can interfere with their ability to be translated,transcribed by polymerases or interact with DNA binding proteins. Inmost cases, the chemical moiety must be removed. Further, the methodsfor removal of these chemical moieties which have detectable physicalproperties often result directly in alteration of the molecule or celldeath.

There are a number of procedures, both complex and simple, which havebeen used to selectively detect and isolate target substrates. Oneprocedure which has revolutionized and greatly accelerated the detectionand identification of nucleic acids is polymerase chain reaction (PCR)technology. The principle concept of PCR is the rapid, large-scaleamplification of unique or even non-unique nucleic acid sequences inbiological samples. Using labeled primers with specific or randomsequences, the genetic code of very small quantities of nucleic acidscan be detected, amplified in number and subsequently characterizedthrough repetitive polymerization events. Although the nucleic acidsformed are new, the sequence of the original sample is maintained andcan be easily determined. As a nucleic acid sequence is the biological.code for the construction of virtually all proteins, the origin,evolutionary age, structure and composition of nearly any biologicalorganism or sample can be determined from knowledge of the sequence. Theprocedure has been proved useful in molecular and evolutionary biology,and has demonstrated applications in the detection, treatment andprevention of diseases and disorders in humans.

PCR technology, although revolutionary, carries with it the samelimitations as many conventional detection and isolation procedures.Label which has been incorporated into primers and ultimately newlyformed nucleic acids must be removed. This process, when possible, isfairly time consuming and often results is modification or destructionof the nucleic acid.

Another example of a process to render a substance specificallydetectable is to use binding molecules which have a particular affinityfor selected other molecules as occurs between binding of an antigen toan antigen-specific antibody. These chemical pairs, sometimes referredto as coupling agents, have been used extensively in detection andisolation procedures. Normally one of the molecules in this pair isimmobilized on an affinity medium such as used in chromatographicpacking material or a magnetic bead and used in the isolation of thetarget molecule. Some of the more useful coupling agents are biotin andavidin or the related protein, streptavidin. These agents have been usedin many separation techniques to facilitate isolation of one componentor another from complex mixtures

Biotin, a water-soluble vitamin, is used extensively in biochemistry andmolecular biology for a variety of purposes including macromoleculardetection, purification and isolation, and in cytochemical staining.Biotin also has important applications in medicine in the areas ofclinical diagnostic assays, tumor imaging and drug delivery, and is usedextensively in the field of affinity cytochemistry for the selectivelabeling of cells, subcellular structures and proteins.

Biotin's utility stems from its ability to bind strongly to thetetrameric protein avidin, found in egg white and the tissues of birds,reptiles and amphibians, or to its chemical cousin, streptavidin,isolated from the bacterium Streptomyces. Typically, biotin or aderivative of biotin is first bound directly to a target molecule, suchas a protein or oligonucleotide, or to a probe using specific chemicallineage. The interaction of the linked biotin with either streptavidinor avidin conjugated to an affinity medium such as magnetic or sepharosebeads is then used in the isolation of the target molecule.Alternatively, the interaction of the covalently linked biotin withavidin or streptavidin conjugated to an enzyme such as horseradishperoxidase (HRP) which catalyzes a chromogenic reaction is used fordetection of the target molecule. Macromolecules that have been isolatedusing biotin-avidin technology are shown in Tables 1 and 2.

TABLE 1 Macromolecules Isolated by Direct Biotinylation BiotinylatedTargets Elution Conditions References Membrane proteins acetate, pH 4 1,2 and glycoproteins Antibodies low pH 3 Enzymes non-physiological 4t-RNA 6 M guanidine-HCl, 5 pH 2.5 rRNA 70% formic acid 6 nucleosomesSS-reduction of 7, 8 cleavable biotin DNA non-physiological 9 1. G. A.Orr, J. Biol. Chem. 256: 761, 1981. 2. C. A. Beard et al., Mol. Biochem.Parasitol. 16: 199, 1985. 3. E. A. Bayer et al., FEBS Lett. 68: 240,1976. 4. C. S. Chandler et al., Biochem. J. 237: 123, 1986. 5. T. R.Broker et al., Nucl. Acids Res. 5: 363, 1978. 6. D. J. Eckermann et al.,Eur. J. Biochem. 82: 225, 1978. 7. M. L. Shimkus et al., Proc. Natl.Acad. Sci. USA 82: 2593, 1986. 8. M. L. Shimkus et al., DNA 5: 247,1986. 9. P. L. Langer et al., Proc. Natl. Acad. Sci. USA 78: 6633, 1981.

TABLE 2 Biological Materials Isolated Using Biotinylated BindingMolecules Binding Target Molecules Molecules Elution Conditions Refs.Glycoproteins conconavalin A 2% SDS 10 Membrane Antigens antibody SDS(boiling) 11 Estrogen Receptor estradiacetate, estradiol 12 InsulinReceptor insulin acetate, pH 5.0 13, 14 biotin Opoid Receptor enkephalinenkephalin 15 Human B antigen selection by FACS 16 lymphocytesLymphocyte monoclonal Mechanical 17, 18, 19 subpopulations antibodyagitation, erythrocyte lysis Plasmid DNA DNA 0.1 M NaOH 20 SpliceosomesRNA 90° C. in SDS 21 Recombinant DNA Cleavable biotin 22 Plasmids Heat,low ionic strength and phenol 10. J. W. Buckie et al., Anal. Biochem.156: 463, 1986. 11. T. V. Updyke et al., J. Immunol. Methods 73: 83,1984. 12. G. Redeulih et al., J. Biol. Chem. 260: 3996, 1985. 13. F. M.Finn et al., Proc. Natl. Acad. Sci. USA 81: 7328, 1984. 14. R. A.Kohanski et al., J. Biol. Chem. 260: 5014, 1985. 15. H. Nakayama et al.,FEBS Lett. 208: 278, 1986. 16. P. Casali et al., Sci. 234: 476, 1986.17. J. Wormmeester et al., J. Immunol. Methods 67: 389, 1984. 18. P. J.Lucas et al., J. Immunol. Methods 99: 123, 1987. 19. R. J. Berenson etal., J. Immunol. Methods 91: 11, 1986. 20. H. Delius et al., Nucl. AcidsRes. 13: 5457, 1985. 21. P. J. Grabowski et al., Sci. 233: 1294, 1986.22. B. Riggs et al., Proc. Natl. Acad. Sci. USA 83: 9591, 1986.

While the utility of biotin continues to grow, there still exists majordrawbacks in the use of biotin-streptavidin technology for manyapplications. This problem stems from the high affinity between biotinand streptavidin, precisely the molecular characteristic which makes itmost useful. Once a target molecule or cell is isolated through thestreptavidin-biotin interaction, release of the target molecule requiresdisruption of this interaction. Dissociation of biotin from streptavidinrequires very harsh conditions such as 6-8 molar (M) guanidinium-HCl, pH1.5. Such conditions also denature, and thereby inactivate, mostproteins and destroy most cells.

For example, a biotin derivative containing a N-hydroxysuccinimide estergroup is commonly used to link biotin through an amide bond to proteinsand nucleic acids. Selective cleavage of this linkage disrupts similarnative chemical bonds in associated molecules. Biotin is also often usedin the isolation of specific cells from a heterogeneous mixture of cellsby binding a biotinylated antibody directed against a characteristiccell surface antigen. The interaction of the biotinylated antibody withstreptavidin-coated magnetic beads or sepharose particles can then beused effectively to isolate target cells. Disruption of theantibody-antigen interaction normally requires exposure of cells toconditions such as low pH or mechanical agitation which are adverse tothe cell's survival. In general recovery of the target in a completelyunmodified form is not possible.

Once the biotinylated DNA is bound to streptavidin it can only bereleased with extreme difficulty. Many diverse methods to remove thestreptavidin molecule have been suggested including digestion byproteinase K (M. Wilchek and E. A. Bayer, Anal. Biochem. 171:1, 1988).Proteinase K also digests nearby proteins and does a fairly poor job ofcompletely digesting the streptavidin. Significant amounts of thestreptavidin molecules remain attached, and further, removal ofstreptavidin does not release the biotin. Further, biotinylated DNAinterferes with subsequent use in a variety of methods includingtransformation of cells and hybridization based assays used fordetection of genetic diseases.

The essentially irreversible binding of biotin and streptavidin is alsoa serious limitation for the performance of multiple or sequentialassays to detect a specific type of biomolecule, macromolecular complex,virus or cell present in a single sample. Normally, only a single assaycan be performed because the enzyme detection system isstreptavidin-based and streptavidin remains firmly bound to thebiotinylated target or target probe. While different chromogenic systemsfor detection are available, they are only of limited applicability insituations where large numbers of probes are needed.

An additional problem in the use of biotin-avidin technology is thepresence of endogenous biotin, either free or complexed to othermolecules, inside the sample to be purified or assayed. In this case,the endogenous biotin can result in the isolation or detection ofnon-target molecules. This can be a particularly severe problem in caseswhere a high signal-to-noise ratio is needed for accurate and sensitivedetection.

To remove biotin from an attached molecule, several chemically cleavablebiotin derivatives have been produced. Immunopure NHS-SS-biotin (PierceChemical; Rockford, Ill.) consists of a biotin molecule linked through adisulfide bond and an N-hydroxysuccinimide ester group that reactsselectively with primary amines. Using this group, NHS-SS-biotin can belinked to a protein and then the biotin portion removed by cleaving thedisulfide bond with thiols. This approach is of limited use since thiolsnormally disrupt native disulfide bonds in proteins. Furthermore, thecleavage still leaves the target cell or molecule modified since thespacer arm portion of the complex is not removed and the cleaving buffermust be eliminated from the sample.

One method for removal of biotin is the use of disulfide-based cleavablebiotins. However, the cleaved molecules possess a reactive sulfhydrylgroup which has a strong tendency to form disulfide bonds with othercomponents of the mixture. Functional activity of these substancescontaining sulfhydryl groups is severely compromised. Typically,activity of such protein is decreased or eliminated and such nucleicacids will no longer hybridize rendering them useless for cloning. Thismethod is also slow and requires the preparation of complex solutions.

An additional limitation of biotin-avidin technology is the difficultyof developing automated systems for the isolation and/or detection oftargets due to the problems of releasing the target from thebiotin-avidin binding complex. This requires addition of specificchemical reagents and careful monitoring of the reactions.

Biotin-avidin technology has been combined with PCR techniques for thedetection and isolation of nucleic acids and specific sequences.However, there still remains a fundamental problem which relates to thedifficulty of removing the incorporated biotin. This is normally notpossible using conventional biotins without irreversibly altering thestructure of the DNA. As discussed, biotinylation can interfere withsubsequent application of biotinylated probes as well as alter theproperties of the PCR product.

PCR products that contain biotinylated nucleotides or primers which arerequired for isolation cannot be used in conjunction with biotinylatedhybridization probes. The presence of biotin on the PCR product causefalse signals from the avidin based enzyme-linked detection system.Biotin incorporation into DNA interferes with strand hybridizationpossibly due to the spacer arms linking the nucleotides to the biotinmolecules. Further, PCR products that are biotinylated are not suitablematerial for cloning. PCR products which contain biotinylatednucleotides are difficult to analyze. Incorporation of biotinylatednucleotides into DNA causes a retardation of mobility during gelelectrophoresis in agarose. This mobility shift renders characterizationof PCR products difficult. As proper DNA-DNA hybridization is the basisfor sensitive and accurate characterization and sensitive assays,biotin-avidin binding systems are seriously disadvantaged.

Other coupling partners which can be used to detect and isolate targetsubstances are cell adhesion molecules (CAMs). One of the wellcharacterized types is the endothelial cell adhesion molecule, LEC-CAM(leukocyte endothelial cell-cell adhesion molecule), now calledselectin. This molecule selectively binds to leukocytes. Its naturalfunction is to facilitate the transport of leukocytes through anendothelial layer of cells such as postcapillary venules to sites ofinflammation or tissue damage. There are many of these adhesionmolecules which have been identified in humans and other mammals thatrange in binding specificity from the very general to the highlyspecific. These include the endothelial cell adhesion ligands ICAM-1,VCAM-1 and ELAM-1, the β-integrins which consists of a family of threeproteins LFA-1, Mac-1, VLA-4, MO-1 and p150/95, carbohydrate bindingCAMs that appear on endothelial cells, platelets, and leukocytes, andthe cadherins, calcium dependent CAMs present on most cells. Attachmentof these molecules or the creation of fused proteins containing adhesiondomains can be used to facilitate isolation and detection of bindingpartners. However, once binding has occurred, complex, expensive andtime consuming biochemical manipulations and sometimes fairly harshchemical treatments are necessary to dissociate the molecules. Further,application of these molecules for general use is limited as bindingpartners must be located for each target of interest

Other coupling partners include nucleic acids and nucleic acid bindingproteins, lipids and lipid binding proteins, and proteins or specificdomains which have a particular affinity for each other. These couplingpartners suffer from similar drawbacks as the biotin-avidin system andthe adhesion molecules.

Another fairly ubiquitous method of detection and isolation is gelelectrophoresis. In this process, a uniform matrix or gel is formed of,for example, polyacrylamide, to which is applied an electric field.Mixtures applied to one end of the gel will migrate through the gelaccording to their size and interaction with the electric field.Mobility is dependent upon the unique characteristics of the substancesuch as conformation, size and charge. Mobilities can be influenced byaltering pore sizes of the gel, such as by formation of a concentrationor pH gradient, or by altering the composition of the buffer (pH, SDS,DOC, glycine, salt). One- and two-dimensional gel electrophoresis arefairly routine procedures in most research laboratories. Targetsubstances can be purified by passage through and/or physical extractionfrom the gel.

Methods for the detection and isolation of targets substances alsoinclude centrifugation techniques such as equilibrium-density-gradientcentrifugation. This process is based on the principal that under highcentrifugal forces, stable gradients win be established in saltsolutions. Mixtures subjected to high speed centrifugation willsegregate individual. components according to their specific densities.Although useful, all of these procedures are more quantitative thanqualitative.

A major advance in detection and isolation methodology was the advent ofliquid chromatography. Chromatography, and in particular columnchromatography, comprises some of the most effective and flexiblepurification methods available. Common to most procedures is the use ofopen cylinders containing a hydrated matrix material. Some of thetypical matrix materials which are presently used, for example, in gelfiltration, affinity chromatography and ion exchange chromatography,include sepharose (bead formed gel prepared from agarose: PharmaciaBiotech; Piscataway, N.J.), sephadex (a bead-formed gel prepared bycross-linking dextran with epichlorohydrin: Pharmacia Biotech;Piscataway, N.J.) and sephacryl (covalently cross-linked allyl dextranwith N,N′-methylene bisacrylamide: Pharmacia Biotech; Piscataway, N.J.).Basically, a heterogenous sample or mixture is applied to the top of thecolumn followed with a suitable buffer. Substances within the mixturedisplay differential migration through the column in relation to othermaterials within the sample and is collected in fractions at the otherend of the column Fractions are individually analyzed for the presenceof target and positive fractions pooled.

Alternatively, target in the sample may selectively bind to the columnmaterial in the presence of buffer a process known as affinitychromatography. After binding, unbound material is removed bycontinuously washing the column with buffer. Target molecules aresubsequently released from the column by application of an elutionbuffer which causes dissociation. Fractions are collected as they eluteoff of the column and collected. In gel-exclusion chromatography, across-linked dextran is utilized as column matrix material.Cross-linking can be varied to alter the effective pore size of thecolumn material and the dextran can be coupled to a wide variety ofchemical moieties to selectively capture target. Ion-exchangechromatography takes advantage of the fact that targets, for exampleproteins, can differ enormously in their affinity for positive ornegative charges on column materials. The affinity of a material for atarget is proportional to the salt concentration of the buffer. Byraising or lowering the salt concentration, it is possible to changeaffinity of target to column material.

Affinity column chromatography makes use of chemical groups that havespecial attraction to the targets of interest. For example, enzymespreferentially bind to certain naturally associated cofactors. Columnmaterials with attached cofactors will selectively bind to such targetenzymes. Enzyme purification becomes a relatively simple andstraightforward matter. In a similar fashion, enzyme-specific antibodiescan be coupled either covalently or non-covalently to a column matrix.The unique affinity of an antibody for its target antigen allows for theselective removal of target from a heterologous mixture of substances.Detection and isolation is again a fairly simple matter.

Two relatively well-established procedures, high-performance liquidchromatography (HPLC) including reverse-phase HPLC and size-exclusionHPLC, and the more recent technique fast-performance liquidchromatography (FPLC) which can handle larger sample volumes than HPLC,is based on standard chromatographic techniques, but using extremelyhigh pressures (5,000 to 10,000 psi and more). Due to the higherpressures, finer column materials can be utilized and separations can beperformed faster and with better resolution.

Although chromatography is an invaluable tool it too has its limits.Materials to be separated must be solubilized into a suitable bufferwhich will not adversely affect the column. Further, substrate mixturesand targets must be capable of passing through a column matrix in areasonable period of time. Although HPLC can sometimes shorten this timeperiod, only small quantities can be detected and the high pressures candamage isolated column material.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides new methodsfor the detection and isolation of molecules from complex mixtures.

One embodiment of the invention is directed to bioreactive agentscomprising a detectable moiety bonded to a photoreactive moiety whereinthe photoreactive moiety contains at least one group capable ofcovalently bonding to a substrate to form a conjugate that can beselectively photocleaved to release said substrate. Detectable moietiesshould have a selectively detectable physical property such asfluorescence, absorption or an ability to specifically bind to acoupling agent such as avidin or streptavidin, antibodies, antigens orbinding proteins. The photoreactive moiety should be capable of formingone or more covalent bonds with a chemical group of a substrate. Thosecovalent bonds may be cleaved or photocleaved with the electromagneticradiation releasing the substrate.

The bioreactive agent may have a chemical structure selected from thegroup consisting of:

wherein X is selected from the group consisting of a halogen, N₂,CH₂-halogen, —N═C═O, —N═C═S, —S—S—R, NC₂H₄, —NC₄H₂O₂, —OH, —NHNH₂,—OP(OR₃)N(R₄)R₅ and —OCO-G, wherein G is selected from the groupconsisting of a halogen, N₃, O-esters and N-amides; R₁, R₂, R₃, R₄ andR₅ are selected from the group consisting of hydrogen, alkyls,substituted alkyls, aryls and substituted aryls, —CF₃, —NO₂, —COOH and—COOR, and may be the same or different; A is a divalent functionalgroup selected from the group consisting of —O—, —S— and —NR₁; Ycomprises one or more polyatomic groups which may be the same ordifferent; V comprises one or more optional monoatomic groups which maybe the same or different; Q comprises an optional spacer moiety; m1 andm2 are integers from 0-5 and may be the same or different; and Dcomprises a detectable moiety which is distinct from R₁-R₅.

Another embodiment of the invention is directed to conjugates comprisinga bioreactive agent photocleavably coupled to a substrate wherein saidagent comprises a detectable moiety bonded to a photoreactive moiety,wherein said conjugate can be selectively cleaved with electromagneticradiation to release said substrate. Suitable substrates which can becoupled to the bioreactive agent include proteins, peptides, aminoacids, amino acid analogs, nucleic acids, nucleosides, nucleotides,lipids, vesicles, detergent micells, cells, virus particles, fattyacids, saccharides, polysaccharides, inorganic molecules, metals, andderivatives and combinations thereof Substrates may be pharmaceuticalagents such as cytokines, immune system modulators, agents of thehematopoietic system, chemotherapeutic agents, radio-isotopes, antigens,anti-neoplastic agents, recombinant proteins, enzymes, PCR products,receptors, hormones, vaccines, haptens, toxins, antibiotics, nascentproteins, cells, synthetic pharmaceuticals and derivatives andcombinations thereof.

Conjugates may have a chemical structure selected from the groupconsisting of:

wherein SUB comprises a substrate; R₁ and R₂ are selected from the groupconsisting of hydrogen, alkyls, substituted alkyls, aryls, substitutedaryls, —CF₃, —NO₂, —COOH and —COOR, and may be the same or different; Ais a divalent functional group selected from the group consisting of—O—, —S— and —NR₁; Y comprises one or more polyatomic groups which maybe the same or different; V comprises one or more optional monoatomicgroups which may be the same or different; Q comprises an optionalspacer moiety; m1 and m2 are integers between 1-5 which can be the sameor different; and D comprises a detectable moiety which is distinct fromR₁ and R₂.

Another embodiment of the invention is directed to pharmaceuticalcompositions comprising the conjugate plus a pharmaceutically acceptablecarrier such as water, an oil, a lipid, a saccharide, a polysaccharide,glycerol, a collagen or a combination thereof. Pharmaceuticals may beused in the prophylaxis and treatments of diseases and disorders inhumans and other mammals.

Another embodiment of the invention is directed to methods for isolatingtargets from a heterologous mixture. Briefly, a conjugate is created bycoupling a bioreactive agent to a substrate by a covalent bond which isselectively cleavable with electromagnetic radiation wherein thebioreactive agent is comprised of a photoreactive moiety bonded to adetectable moiety. The conjugate is contacted to the heterologousmixture to couple substrate to one or more targets. The coupledconjugate is separated from the mixture and treated with electromagneticradiation to release the substrate, and the targets isolated. Thismethod can be used to isolate targets such as immune system modulators,cytokines, agents of the hematopoietic system, proteins, hormones, geneproducts, antigens, cells, toxins, bacteria, membrane vesicles, virusparticles, and combinations thereof from heterologous mixtures such asbiological samples, proteinaceous compositions, nucleic acids, biomass,immortalized cell cultures, primary cell cultures, vesicles, animalmodels, mammals, cellular and cell membrane extracts, cells in vivo andcombinations thereof

Another embodiment of the invention is directed to target moleculesisolated by the above methods which may be used in pharmaceuticalcompositions or other compositions and mixtures for industrialapplications.

Another embodiment of the invention is directed to methods for isolatingtargets from a heterologous mixture. A conjugate is created comprising abioreactive agent coupled to a substrate by a covalent bond which isselectively cleavable with electromagnetic radiation, wherein saidbioreactive agent is comprised of a photoreactive moiety bonded to adetectable moiety and the substrate is a precursor of the target. Theconjugate is contacted with the heterologous mixture to incorporatesubstrate into targets. The incorporated conjugate is separated from themixture, treated with electromagnetic radiation to release thesubstrate, and the targets isolated. This method is useful for thedetection and isolation of nascent proteins, nucleic acids and otherbiological substances.

Another embodiment of the invention is directed to methods for isolatingtargets from a heterologous mixture. A conjugate is created which iscomprised of a bioreactive agent coupled to a receptor by a covalentbond which is selectively cleavable with electromagnetic radiation,wherein said bioreactive agent is comprised of a photoreactive moietybonded to a detectable moiety. The conjugate is contacted with theheterologous mixture to couple receptor to targets and the coupledreceptor-targets separated from the mixture. The separated conjugate istreated with electromagnetic radiation to release the receptor and thetargets isolated.

Another embodiment of the invention is directed to methods for isolatingtarget cells from a heterologous mixture. A conjugate is createdcomprising a bioreactive agent coupled to a cell receptor by a covalentbond which is selectively cleavable with electromagnetic radiation,wherein the bioreactive agent is comprised of a photoreactive moietybonded to a detectable moiety. The conjugate is contacted with theheterologous mixture to couple receptor to target cells. The coupledconjugate is separated from the mixture and treated with electromagneticradiation to release the substrate. Target cells are then easilyisolated such as by automation.

Another embodiment of the invention is directed to methods for creatinga photocleavable oligonucleotide. A conjugate is created comprising abioreactive agent coupled to a phosphoramidite which may be apurine-phosphoramidite or a pyrimidine-phosphoramidite. Theoligonucleotide is synthesized using photocleavable phosphoramidites.The process can be performed manually or automated to be carried out byan oligonucleotide synthesizer.

Another embodiment of the invention is directed to methods fordetermining an in vivo half-life of a pharmaceutical in a patient. Aconjugate is formed by coupling the pharmaceutical to a bioreactiveagent with a covalent bond that can be selectively cleaved withelectromagnetic radiation, wherein said bioreactive agent comprises aphotoreactive moiety bonded to a detectable moiety. The conjugate isadministered to the patient and at least two or more biological samplesare removed from the patient at various times after administration ofthe conjugate. The samples are treated with electromagnetic radiation torelease the pharmaceutical from the bioreactive agent and the amount ofthe bioreactive agent in the biological samples determined. The in vivohalf-life of the pharmaceutical can be determined.

Another embodiment of the invention is directed to methods for thecontrolled release of a substrate into a medium. A conjugate comprisedof a bioreactive agent coupled to the substrate by a covalent bond whichcan be selectively cleaved with electromagnetic radiation is createdwherein the bioreactive agent is comprised of a detectable moiety bondedto a photoreactive moiety. The conjugate is bound to a surface of anarticle which is placed into the medium. The surface of the article isexposed to a measured amount of electromagnetic radiation for thecontrolled release of the substrate into the medium.

Another embodiment of the invention is directed to methods for detectinga target molecule in a heterologous mixture. A conjugate is formed bycoupling a substrate to a bioreactive agent with a covalent bond that isselectively cleavable with electromagnetic radiation, wherein thebioreactive agent is comprised of a detectable moiety bonded to aphotoreactive moiety. The conjugate is contacted with the heterologousmixture to couple substrate to one or more target molecules. Uncoupledconjugates are removed and the coupled conjugates are treated withelectromagnetic radiation to release the detectable moiety. The releaseddetectable moiety can now be easily detected.

Another embodiment of the invention is directed to methods for detectinga target molecule in a heterologous mixture. A conjugate, comprising asubstrate coupled to a bioreactive agent, is formed and contacted with aheterologous mixture to couple a conjugate to one or more targetmolecules. Uncoupled conjugates are removed and the coupled conjugatesare treated with electromagnetic radiation to release substrate.Released substrate is detected and can be further isolated.

Another embodiment of the invention is directed to methods for theisolation of a PCR product. A bioreactive agent is conjugated to one ormore oligonucleotide primers with a covalent bond that is selectivelycleavable with electromagnetic radiation, wherein the bioreactive agentis comprised of a detectable moiety bonded to a photoreactive moiety. Anucleic acid sequence is PCR amplified with the conjugated primers. Theamplified sequences are isolated and subsequently treated withelectromagnetic radiation to release the bioreactive agent.

Another embodiment of the invention is directed to methods for treatinga disorder by the controlled release of a therapeutic agent at aselected site. A conjugate is formed by bonding a bioreactive agent tothe therapeutic agent with a bond that is selectively cleavable withelectromagnetic radiation, wherein the bioreactive agent is comprised ofa directable moiety bonded to a photoreactive moiety wherein thedirectable moiety has an affinity for the selected site. The conjugateis administered to a patient having the disorder. The selected site issubjected to a measured amount of electromagnetic radiation for thecontrolled release of the therapeutic agent to treat the disorder.

Another embodiment of the invention is directed to kits for detecting adisorder in biological samples containing conjugates comprised of abioreactive agent covalently bonded to a diagnostic agent having anaffinity for an indicator of the disorder in the biological sample,wherein the covalent bond is selectively cleavable with electromagneticradiation.

Another embodiment of the invention is directed to kits comprising abioreactive agent covalently bonded to an oligonucleotide. Thephotocleavable oligonucleotide may be double-stranded or single-strandedand may possess restriction enzyme recognition sites useful in cloningand other procedures in molecular biology.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description or may be learned from the practice of theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1 Examples of photocleavable agents.

FIG. 2 Photocleavable biotins with various photoreactive moieties.

FIG. 3 Schematic representation of photocleavable biotin

FIG. 4 Mechanism of photocleavage in 2-nitrobenzyl-based systems.

FIG. 5 Synthesis of photocleavable biotin.

FIG. 6 Chemical variations of photocleavable agents.

FIG. 7 Photolysis of PCBs.

FIG. 8 PCB conjugates.

FIG. 9 Possible amino acid linkages of PCB.

FIG. 10 (A) Aminoacylation of tRNA, and (B) a comparison betweenenzymatic 5 and chemical aminoacylation.

FIG. 11 The four basic steps in the isolation of pure substrate usingPCB.

FIG. 12 Method for the detection of target protein and its antibodyusing PCB.

FIG. 13 PCB-phosphoramidites and PCB-nucleotides.

FIG. 14 Two methods (A and B) for the isolation of PCR-product usingPCB.

FIG. 15 Comparison of methods for the construction of a cDNA library (A)with PCB and (B) without PCB. SEQ. ID. No.: 1

FIG. 16 PCB lipids.

FIG. 17 Immunoselective cell separation using PCB.

FIG. 18 Synthesis of photocleavable biotin-NHS ester, compound 18.

FIG. 19 Synthesis of photocleavable biotin-NHS ester, compound 25.

FIG. 20 Method for sequential ELISA using PCB.

FIG. 21 Synthesis of photocleavable coumarin.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to agents and conjugates used in the detection and isolation oftargets such as chemicals, macromolecules, cells and any identifiablesubstance from a mixture. Agents comprise a detectable moiety bound to aphotoreactive moiety. Conjugates comprise agents which are coupled tosubstrates by one or more covalent bonds which, by the presence of thephotoreactive moiety, are selectively cleavable with electromagneticradiation. The invention is also directed to methods for the isolationand detection of targets using these agents and conjugates, to kitswhich utilize these methods for the detection of diseases and disordersin patients, and to methods for the detection and isolation of nearlyany substance from a heterologous mixture.

There are many methods currently available for the detection andisolation of a desired substance or target from a complex mixture. Mostof these methods require the specific labeling of the substance ortarget to be detected, detection of that label and subsequent removal oflabel from target. Although straightforward, current detection andisolation methodologies possess a number of problems. For example, it isoften difficult to specifically attach target with label. Affinity oflabel for target may be low, suitable points of attachment may not beavailable on specific substances and the label and the target may simplybe chemically or physically incompatible. In addition, label may hinderor completely destroy the functional activity of the target frustratingthe purpose of isolation. Isolated targets are unavoidably contaminatedor inactivated due to the presence of a toxic or damaging label. Atypical example of this sort of problem is the isolation of cells boundwith biotin after selection by coupling to streptavidin. The powerfulaffinity of biotin for streptavidin makes the isolation procedurerelatively straightforward and specific, however, the isolated cells areoften dead and dying due to the toxic effects of the coupling agents orthe harsh isolation procedures. This is also true when attempting toisolate active proteins from biological samples and other complexmixtures for later use. The presence of label may denature or render theprotein product inactive or simply unacceptable for in vivo use undercurrent FDA standards and guidelines. Removal of the agent sometimesovercomes these problems, however, methods to separate and remove labelfrom target are generally rather harsh, take a significant amount oftime, effort and expense, and, for the most part, result in fairly lowyields of the final product. Viability and functional activity of thetarget is often severely impaired and is often destroyed.

The invention overcomes these problems by providing detectable,bioreactive agents which can detect and isolate targets. Agents of theinvention comprise a detectable moiety and a photoreactive moiety, andcan be covalently coupled to a variety of target substrates. A covalentbond between agent and substrate can be created from a wide variety ofchemical moieties including amines, hydroxyls, imidazoles, aldehydes,carboxylic acids, esters and thiols. Agent-substrate combinations arereferred to herein as conjugates. Through the presence of the detectablemoiety, conjugates can be quickly and accurately detected and targetisolated. Further, these conjugates are selectively cleavable whichprovides unique advantages in isolation procedures. Substrate can beseparated from agent quickly and efficiently. Complex technicalprocedures and highly trained experts are not required. New attachmentand separation procedures do not need to be developed for every newtarget to be isolated. Following isolation, it is a relatively simplematter to treat the conjugate with electromagnetic radiation and releasethe substrate. Released substrate is preferably functionally active andstructurally unaltered. Nevertheless, minor chemical alterations in thestructure may occur depending on the point of attachment. It isgenerally preferred that such alterations not effect functionalactivity. However, when functional activity does not need to bepreserved, such changes are of no considerations and may even be usefulto identify and distinguish targets isolated by methods of theinvention.

Targets, as referred to herein, are those substances being identified,characterized or isolated using the agents, conjugates and methods ofthe invention. Substrates, as referred to herein, are those substanceswhich are covalently attached to the bioreactive agent. Substrates mayalso be referred to as targets when the target being identifiedspecifically binds to the bioreactive agent.

One embodiment of the invention is directed to a bioreactive agentcomprising a detectable moiety bonded to a photoreactive moiety whereinthe photoreactive moiety contains at least one group capable ofcovalently bonding to a substrate to form a conjugate. The resultingconjugate can be selectively cleaved to release said substrate or,alternatively, to release any chemical group or agent of the conjugate.Cleavage, as referred to herein, is by photocleavage or a cleavage eventtriggered by the application of radiation to the conjugate. Theradiation applied may comprise one or more wavelengths from theelectromagnetic spectrum including x-rays (about 0.1 nm to about 10.0nm; or about 10¹⁸ to about 10¹⁶ Hz), ultraviolet (UV) rays (about 10.0nm to about 380 nm; or about 8×10¹⁶ to about 10¹⁵ Hz), visible light(about 380 nm to about 750 nm; or about 8×10¹⁴ to about 4×10¹⁴ Hz),infrared light (about 750 nm to about 0.1 cm; or about 4×10¹⁴ to about5×10¹¹ Hz), microwaves (about 0.1 cm to about 100 cm; or about 10⁸ toabout 5×10¹¹ Hz), and radio waves (about 100 cm to about 10⁴ m; or about10⁴ to about 10⁸ Hz). Multiple forms of radiation may also be appliedsimultaneously, in combination or coordinated in a step-wise fashion.Radiation exposure may be constant over a period of seconds, minutes orhours, or varied with pulses at predetermined intervals.

Typically, the radiation source is placed at a specified distance fromthe conjugate to be irradiated. That distance may be empiricallydetermined or calculated from the energy loss produced between thesource and the target and the amount of energy emitted by the source.Conjugate may be in solution or attached to a solid support which may bea type of glass, ceramic, polymer or semiconductor surfaces. Typicalsolid supports are nitrocellulose membranes, agarose beads, magneticbeads coated with streptavidin, semiconductor surfaces and resins.Preferably, the radiation applied is UV, visible or IR radiation of thewavelength between about 200 nm to about 1,000 nm, more preferablybetween about 260 nm to about 600 nm, and more preferably between about300 nm to about 500 nm. Radiation is administered continuously or aspulses for hours, minutes or seconds, and preferably for the shortestamount of time possible to minimize any risk of damage to the substrateand for convenience. Radiation may be administered for less than aboutone hour, preferably less for than about 30 minutes, more preferably forless than about ten minutes, and still more preferably for less thanabout one minute. Visible, UV and IR radiation are also preferred as allthree of these forms of radiation can be conveniently and inexpensivelygenerated from commercially available sources.

The power density or intensity of light per area necessary toselectively cleave the covalent bond is very small which makes thephotocleavable process practical. Maximization of efficiency alsominimizes exposure time necessary to achieve selective cleavage andprovide a minimum of undesirable background effects.

One part of the bioreactive agent is the detectable moiety. Thedetectable moiety is a chemical group, structure or compound thatpossesses a specifically identifiable physical property which can bedistinguished from the physical properties of other chemicals present inthe heterologous mixture. Fluorescence, phosphorescence and luminescenceincluding electroluminescence, chemiluminescence and bioluminescence areall detectable physical properties not found in most substances, butknown to occur or to be inducible in others. For example, reactivederivatives of dansyl, coumarins, rhodamine and fluorescein are allinherently fluorescent when excited with light of a specific wavelengthand can be specifically bound or attached to other substances. Coumarinhas a high fluorescent quantum yield, higher than even a dansyl moiety,and facilitates detection where very low levels of target that are beingsought. Coumarin is structurally similar to tryptophan, which can beuseful in for example in the translation of nascent proteins withnon-native amino acids. It may also be useful to combine certaindetectable moieties to facilitate detection or isolation. Preferably thedetectable moiety is a fluorescent compound and the preferredfluorescent compounds are listed in Table 3, all of which arecommercially available (Sigma Chemical; St. Louis, Mo.).

TABLE 3 Fluorescent Labeling Compounds4-acetamido-4′-isothiocyanatostilbene-2-2′-disulfonic acid7-amino-4-methylcoumarin (AMC) 7-amino-4-trifluoromethylcoumarinN-(4-anilino-1-naphthyl) maleimide 4′,6-diamidino-2-phenylindole (DAPI)5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)4,4′-dilsothiocyanatostilbene-2,2′-disulfonic acid tetramethylrhodamineisothiocyanate (TRITC) quinolizino fluorescein isothiocyanate (QFITC)dansyl chloride eosin isothiocyanate erythrosin B fluorescaminefluorescene fluorescein derivatives 4-methylumbelliferoneo-phthaldialdehyde rhodamine B rhodamine B derivatives rhodamine 6Grhodamine 123 sulforhodamine B sulforhodamine 101 sulforhodamine 101acid chloride

Luminescence can also be induced in certain chemicals referred to asluciferins. Energetic molecules such as ATP supply chemical energy forcatalytic activities of luciferase enzymes causing the luciferins toemit light. Reagents for both the bacterial luciferase system (Vibrioharveyi or V. fischeri) and the firefly luciferase system (Photinuspyralis) are available from a variety of commercial sources (e.g. SigmaChem. Co; St. Louis, Mo.).

Preferably, the luminescent agent has a high quantum yield offluorescence at a wavelength of excitation different from that used toperform the photocleavage. Upon excitation at such wavelengths, theagent is detectable at low concentrations either visually or usingconventional luminescence detectors and fluorescence spectrometers.Electroluminescence, produced by agents such as ruthenium chelates andtheir derivatives, or agents that possess nitroxide moieties and similarderivatives are preferred when extreme sensitivity is desired (J.DiCesare et al., BioTechniques 15:152-59, 1993). These agents aredetectable at the femtomolar ranges and below.

Application of an electric field will also induce a detectable responsein certain chemicals' due to a net electric charge which induces thesubstance to migrate in an electric field. Magnetism may also be adetectable property if a magnetized substance such as iron or anothermagnetized metal is or is associated with the detectable moiety.

Other forms of detectable physical properties include an identifiableelectrical polarizability, electron spin resonance and Raman scattering.Agents may also undergo a chemical, biochemical, enzymatic,electrochemical or photochemical reaction such as a color change inresponse to external electromagnetic fields or the introduction of othersubstances. Such electromagnetic fields and substances may be a catalystor another reactant molecule that allows for detection of thebioreactive agent or transforms the agent into a detectable moiety.

All of these labeling agents can be specifically detected using theappropriate detector or detection system such as a spectrometer orelectrophoretic or chromatographic systems. At times, it may bepreferable to have a visually discernable detection system such as onethat will trigger a photoelectric cell or one that can be detected andrecorded manually. Spectrometers including absorption and fluorescencespectrometers are very sensitive detectors of specific energy ofabsorptions or emissions from many detectable moieties. Detection andsorting of target may be automated as in the case of fluorescenceactivated cell sorters (FACS) which detect and isolate cells thatpossess a fluorescent label. Targets may be detected and sorted manuallyas can be done quite simply with magnetized conjugates using a magnet.

Additional physical properties which can be easily and accuratelydetected include chromaticism (e.g. violet=about 400-430 nm, blue=about450-500 nm, green=about 550 nm, yellow=about 600 nm, orange=about 650nm, red=about 700-750 nm), electromagnetic absorbance, enzyme activityor the ability to specifically bind with a coupling agent. Usefulcoupling agents include biotin, avidin, streptavidin, nucleic acids,nucleic acid and lipid binding proteins, haptens, antibodies, receptors,carbohydrates, immunogenic molecules, and derivatives and combinationsthereof. The detectable moiety may have a combination of theseproperties allowing its selection from a wide variety of backgroundmaterials. Some examples of the chemical structures of photocleavableagents of the invention are depicted in FIG. 1.

Another preferred detectable moiety is a coupling agent and thepreferred coupling agent is biotin or a biotin derivative.Biotin-containing bioreactive agents are referred to herein asphotocleavable biotins or PCBs. The binding between the egg-whiteprotein avidin, a tetrameric protein found in avian eggs with the watersoluble vitamin, biotin, is one of the strongest interactions known inbiology having an association constant (K_(a)) of about 10¹⁵M⁻¹,exceeding that of antibody-antigen interactions (M. Wilchek and E. A.Bayer, Methods Enzymol. 184, 1990). The bacterial counterpart to avidinis streptavidin, found in Streptomyces avidinii, which is slightly morespecific for biotin than avidin. This strong interaction, along with theability to covalently link biotin to a variety of substrates includingproteins, nucleic acids, lipids, and receptor ligands such asneuropeptides and hormones, has resulted in a vast array of uses forthese coupling agents all of which can be improved or enhanced with theuse of PCB.

A wide variety of biotinyl moieties can be used to form a PCB molecule.Biotin (C₁₀H₁₆N₂O₃S) has a molecular weight of 244.31 daltons and iscomprised of a ring linked to an alkyl chain terminated by a carboxylgroup. Numerous modifications can be made to the biotin moiety whichinvolve changes in the ring, spacer arm and terminating group, all ofwhich still exhibit a high affinity for streptavidin, avidin and theirderivatives. Examples of photocleavable biotins that can be designedbased on various photoreactive moieties are depicted in FIG. 2.

The detection and isolation of chemical, biochemical and biologicalmaterials using the interaction between biotin and streptavidin isnormally based on the immobilization of avidin or streptavidin to asurface (e.g. membranes, gels, filters, microtiter wells, magneticbeads). To that surface is applied a solution containing biotin coupledto targets which then bind to the streptavidin-coated surface.Biotin-containing target molecules can be isolated and non-biotinylatedcomponents washed away. Alternatively, biotinylated target molecules canbe separated from a heterogeneous mixture using streptavidin-containingaffinity columns. Biotinylated macromolecules including nucleic acids(DNA or RNA), proteins and protein-containing complexes, and even cellswhose surface has been biotinylated or bound to a biotinylated antibodycan be detected and isolated with these techniques.

As stated, biotin can be coupled to a wide variety of moleculesincluding proteins, carbohydrates and nucleic acids. The availability ofbiotin derivatives has expanded this range even further. For example,biotin derivatives have been prepared with functionalities which arereactive towards amines, phenols, imidazoles, aldehydes, carboxylicacids and thiols. Biotin can also be incorporated into proteins, DNA andRNA by first attaching the biotin to building blocks of macromoleculessuch as amino acid or nucleotides which can be directly attached tothese molecules or incorporated during their synthesis by chemical orenzymatic means.

Unlike conventional biotins, photocleavable biotins enable one torelease or elute the bound substrate from the immobilized avidin,streptavidin or their derivatives in a completely unmodified form. Thisis extremely useful and an important improvement over existing biotinsfor a number of reasons. Biotinylation of the target material can impedeits subsequent use or characterization. Biotinylation of a protein canalter its activity, electrophoretic mobility, ability to bind asubstrate, antigenicity, ability to reconstitute into a native form andability to form multisubunit complexes. In contrast, usingphotocleavable biotin, once the biotin is photocleaved from the proteinor protein/binding complex, all the native properties and function willbe restored to its native form for further use and characterization.Listed in Table 4 are some of the substrates to which a photocleavableagent such as PCB can be linked.

TABLE 4 Chemical linkage of Photocleavable Biotins with differentmolecules Resulting linkage Molecule or Functional Group Reactive moietyand reaction Assemblage on the Molecule on the PCB conditions Aminoacids, Amino group or NHS-ester Amide linkage proteins, enzymes R—NH₂ orantibodies Amino acids, R—OH chloroformate Ester linkage proteins,enzymes carboxylic acid or antibodies Amino acids, R—COOH Reaction withEster linkage proteins, enzymes parent alcohol or antibodies (DCCcoupling) Nucleotides, Aromatic amines chloroformate Amides RNA or DNAmolecules Carbohydrates Sugar hydroxy chloroformate Ester linkage RNAfor R—OH ribonucleotides Nucleotide Phosphate groups diazoethanePhosphate ester phosphoramidites Lipids/ R—NH₂ Chloroformate amidePhosphatidyl NHS-ester serine Carbohydrates Sugar hydroxyl ChloroformateEster linkage

There are a number of chemical moieties available in bioreactive agentsand conjugates of the invention. For example, an NHS-ester functionalityintroduced in PCB is highly reactive and can selectively react withaliphatic amino groups that are present in proteins. Another example isa phosphoramidite moiety which is highly reactive and can selectivelyreact with hydroxyl groups of nucleic acids. In cases where chemicalmoieties like carboxyl (—COOH) or phosphate groups need modification, aprecursor of PCB in the form of the parent alcohol can be used to formappropriate ester-type linkages. These derivatives can be chemicallylinked to a variety of macromolecules and molecular components includingamino acids, nucleotides, proteins and polypeptides, nucleic acids (DNA,RNA, PNA), lipids, hormones and molecules which function as ligands forreceptors.

The application of biotin-avidin technology for the detection andisolation of chemical and biological materials has also been broadenedby the use of binding molecules which are first biotinylated and thenallowed to selectively interact with the target molecule to be isolated.Isolation of the target molecule or cell is facilitated by the bindingof the biotinylated binding-complex to the streptavidin-containingcolumn or streptavidin-coated magnetic beads. Binding molecules includeantibodies which selectively bind to specific antigens, DNA probes whichselectively bind to specific DNA sequences and ligands which selectivelybind to specific receptors. This approach has been used to isolate awide variety of macromolecules and cells (Tables 2 and 3). However, suchisolation methods require that the biotinylated target be released fromthe bound streptavidin. Disruption of this bond typically requiresnon-physiological conditions such as low pH and high concentration ofguanidinium-HCl which is usually damaging for the target molecule orcell. Even after disruption of the streptavidin-biotin interaction, thetarget or binding molecule remains partially or completely biotinylatedwhich can interfere with later uses. Further, elution conditions arenon-physiological and can also be disruptive to the target molecule orcell. In contrast, using photocleavable biotins substrate can be quicklyand easily cleaved from biotin with little to no effect on substrateconformation or activity.

The use of PCB in any of the usual detection and purificationprocedures, including those discussed above, represents a significantsavings of time, energy and ultimately cost. In addition, a variety ofderivatives of avidin and streptavidin are commercially available whichhave been modified through chemical or genetic means. These samederivatives can be used with PCB. One example is ImmunoPure NeutrAvidinsold commercially (Pierce Chemical; Rockford, Ill). This protein is amodified avidin derivative which has been deglycosylated and does notcontain the RYD domain that serves as a universal recognition sequencefor many cell receptors. Non-specific adsorption to other proteins andcell surfaces is greatly reduced.

The major molecular elements of a photocleavable biotin (PCB) are aphotoreactive moiety and a biotinyl moiety which constitutes thedetectable moiety (FIG. 3). The photoreactive moiety and the biotinylmoiety are linked together with a spacer arm to form the PCB molecule.The photoreactive moiety contains a five or six membered ringderivatized with functionalities represented by X, Y and A-C(O)-G,wherein X allows linkage of PCB to the biomolecular substrate. In thepreferred embodiment, Y represents a substitution pattern on a phenylring containing one or more substituents such as nitro or alkoxy groups.The functionality W represents the group that allows linkage of thecross-linker moiety to the photoreactive moiety. The purpose of thespacer arm is to increase the access of the biotin moiety for effectiveinteraction with streptavidin, and thus, increase the bindingefficiency. Typically these can be constructed using either long alkylchains or using multiple repeat units of caproyl moieties linked viaamide linkages.

Choice of photolabile group, spacer arm and the biotinyl moiety dependson the target substrate including amino acids, proteins, antibodies,nucleotides, DNA or RNA, lipids, carbohydrates and cells to which thephotocleavable biotin is. to be attached. It also depends on the exactconditions for photocleavage and the desired interaction between thebiotinyl moiety and streptavidin, avidin or their derivatives. Some ofthe various choices for the photolabile group and linker arms for PCBare shown in FIG. 2.

Additional types of coupling agents include antibodies, antibodyfragments and antigens. Antibodies have the advantage that they can bindto their respective antigen with great specificity. Substrates which areantigens can be detected by their ability to specifically bind toavailable antibodies or to antibodies which can be easily created.Useful antibodies or antibody fragments may be monoclonal or polyclonaland are preferable of the class IgG, but may also be IgM, IgA, IgD orIgE. Other preferred detectable moieties include nucleic acids. Shortsequences of RNA or DNA or oligonucleotides, preferably less than aboutthirty nucleotides in length and as short as four to ten nucleotides,can be detected by their ability to specifically hybridize with acomplementary nucleic acid and detected directly or indirectly using PCRwhich greatly amplifies a specific sequence that is subsequentlydetected. In a similar fashion, binding proteins and receptor-ligandcombinations are also useful as detectable labels.

The second component of the bioreactive agent is the photoreactivemoiety. The photoreactive moiety is a chemical moiety capable of formingone or more covalent bonds with a substrate which can be cleaved withelectromagnetic radiation. These bonds may be formed with a chemicalgroup on the substrate such as, for example, an amine, phenol,imidazole, aldehyde, carboxylic acid or thiol. The photoreactive agentis a substituted aromatic ring containing at least one polyatomic groupand, optionally, one or more monoatomic groups. The aromatic ring ispreferably a five or six-membered ring. The substitutions comprise thepolyatomic and optional monoatomic groups. The polyatomic group impartselectron channeling properties to attract or repel electrons to certainlocations within the chemical structure, thereby creating orestablishing the conditions to create the selectively cleavable covalentbonds. Some monoatomic groups such as halides can adjust the frequencyor wavelength of the electromagnetic radiation which will inducecleavage. As such, monoatomic groups fine tune the cleavage event tosensitize conjugates to predetermined frequencies or intensities ofradiation.

One class of photoreactive moieties are 2-nitrobenzyl derivatives. Intheir ground state, 2-nitrobenzyl-based agents and conjugates have anintramolecular hydrogen bond between benzylic hydrogen and the orthonitro group (—CH—O₂N) (B. Brzezinski et al., J. Chem. Soc. Perkin.Trans. 2:2257-61, 1992). Upon illumination with wavelengths of greaterthan 300 nm, these chemical compounds transition to an excited state.Proton transfer reaction from benzylic carbon to the oxygen in nitrogroup takes place which is followed by electron rearrangement (FIG. 4).This reaction results in the formation of a transient species called anaci-nitro ion which is in a rapid equilibrium with a cyclic form. In thecyclic intermediate, electron rearrangement and oxygen transfer fromnitrogen to benzylic carbon takes place resulting in the formation of2-nitroso derivatives and release of a substrate which is a good leavinggroup (J. A. McCray et al., Annu. Rev. Biophys. Chem. 18:239-70, 1989).

Chemical synthesis of PCB NHS-ester involves three principal steps: (1)Generation of the photoreactive moiety, for example,5-methyl-2-nitroacetophenone. (2) Generation of a suitable amino groupand attachment to biotin containing spacer. (3) Generation of hydroxylgroups and derivatization as N-hydroxysuccinimidyl carbonate(NHS-ester). These steps are schematically represented in FIG. 5.

Bioreactive agents can also be synthesized based on other photoreactivemoieties. Chemical syntheses of two other classes of photocleavablemoieties 3,5-dimethoxybenzyl and 2-nitrobenzenesulfenyl (FIG. 2) can becarried out using similar synthesis strategies. These 2-nitrobenzylgroups all contain a benzylic carbon-hydrogen bond ortho to a nitrogroup, which is necessary for their photolability. In the developmentsof these photolabile groups as protecting groups, difficulties wereencountered as the subsequent reactions of these carbonyl compoundsresulted in formation of coupled azo compounds, which act as internallight filters (V. N. R. Pillai, Synthesis 1, 1980). These complicationswere overcome in the present invention with the use of α-substituted,o-nitrobenzyl compounds. Bioreactive agents of the invention that form adetectable photocleavable conjugate can, for example, be represented bythe formula:

wherein X is selected from the group consisting of a halogen, N₂,CH₂-halogen, —N═C═O, —N═C═S, —S—S—R, NC₂H₄, —NC₄H₂O₂, —OH, —NHNH₂,—OP(OR₃)N(R₄)R₅ and —OCO-G wherein G is selected from the groupconsisting of a halogen, N₃, O-esters and N-amides; R₁, R₂, R₃, R₄ andR₅ are selected from the group consisting of hydrogen, alkyls,substituted alkyls, aryls and substituted aryls, —CF₃, —NO₂, —COOH and—COOR, and may be the same or different; A is a divalent functionalgroup selected from the group consisting of —O—, —S— and —NR₁; Ycomprises one or more polyatomic groups which may be the same ordifferent; V comprises one or more optional monoatomic groups which maybe the same or different; Q comprises an optional spacer moiety; m1 andm2 are integers from 0-5 and may be the same or different; and Dcomprises a selectively detectable moiety which is distinct from R₁-R₅.The O-ester may be cyanomethyl, o and p nitrophenyl, 2,4-dinitrophenyl,2,4,5-trichlorophenyl, pentachlorophenyl, pentafluorophenyl,N-hydroxyphthalimidyl, N-hydroxysuccinimidyl, 1-hydroxypiperidinyl,5-chloro-8-hydroxy-quinolyl, 1-hydroxybenzotriazolyl,3,4-dihydro-4-oxobenzotriazin-3-yl (DHBT),2,3-dihydro-2,5-diphenyl-3-oxo-thiophen-1,1-dioxide-4-yl (TDO),1,2-benzisoxasolyl, 2-hydroxypyridyl or derivatives or combinationsthereof. The N-amide is an imidazolyl, benzimidazolyl, benzisoxazolyl,3,5-dioxo-4-methyl-1,2,4-oxadiezolidinyl or derivatives or combinationsthereof.

Polyatomic groups can be attached to the aromatic ring include nitrogroups (—NO₂), sulfoxide groups such as (—SO₃), alkyl groups such asmethyl (—CH₃) and ethyl groups (—CH₂CH₃), alkoxyl groups such as(—OCH₃), and derivatives and combinations thereof. Useful monoatomicgroups are halides such as chloride (—Cl), fluoride (—F), iodide (—I),bromide (—Br), and hydrogen (—H). These groups may be placed in any ofthe available positions around the ring. Selection and placement of thepolyatomic and monoatomic groups may influence the wavelength ofelectromagnetic radiation required to induce cleavage and the period oftime that radiation must be applied to induce efficient cleavage. Thechemical moieties at R₁, R₂, R₃, R₄ and/or R₅ may also influence thephotoreaction.

It is sometimes useful to include in the agent a spacer moiety bondedbetween the photoreactive moiety and the detectable moiety. The presenceof the spacer can be advantageous sterically for substrate binding. Thespacer moiety (Q) may comprise a branched or straight chain hydrocarbon,a polymeric carbohydrate, or a derivative or combination thereof. Thepreferred spacer moiety is represented by the formula:

wherein W and W′ are each selected from the group consisting of —C(O)—,—C(O)—NH—, —HN—C(O)—, —NH—, —O—, —S— and —CH₂—, and may be the same ordifferent; and n1 and n2 are integers from 0-10 which can be the same ordifferent and if either n1 or n2 is zero, then W and W′ are optional.Examples of the chemical structure of bioreactive agents are depicted inFIG. 6.

Another embodiment of the invention is directed to photocleavableconjugates comprising bioreactive agents photocleavably coupled tosubstrates. Conjugates have the property that they can be selectivelycleaved with electromagnetic radiation to release the substrate.Substrates are those chemicals, macromolecules, cells and othersubstances which are or can be used to detect and isolate targets.Substrates that are selectively cleaved from conjugates may be modifiedby photocleavage, but still functionally active, or may be released fromthe conjugate completely unmodified by photocleavage. Substrates may becoupled with agents, uncoupled and recoupled to new agents at will.

Useful substrates are any chemical, macromolecule or cell that can beattached to a bioreactive agent. Examples of useful substrates includeproteins, peptides, amino adds, amino acid analogs, nucleic acids,nucleosides, nucleotides, lipids, vesicles, detergent micells, cells,virus particles, fatty acids, saccharides, polysaccharides, inorganicmolecules and metals. Substrates may also comprise derivatives andcombinations of these substances such as fusion proteins,protein-carbohydrate complexes and organo-metallic compounds. Substratesmay also be pharmaceutical agents such as cytokines, immune systemmodulators, agents of the hematopoietic system, recombinant proteins,chemotherapeutic agents, radio-isotopes, antigens, anti-neoplasticagents, enzymes, PCR products, receptors, hormones, vaccines, haptens,toxins, antibiotics, nascent proteins, synthetic pharmaceuticals andderivatives and combinations thereof.

Substrates may be targets or part of the targets such as an amino acidin the synthesis of nascent polypeptide chains wherein substrates may beamino acid or amino acid derivative which becomes incorporated into thegrowing peptide chain. Substrates may also be nucleotides or nucleotidederivatives as precursors in the synthesis of a nucleic acid. Constructsuseful in creating synthetic oligonucleotide conjugates may containphosphoramidites or derivatives of DATP, dCTP, dTTP and dGTP, and alsoATP, CTP, UTP and GTP. Resulting nucleic acid-conjugates can be used inPCR technologies, antisense therapy, and prophylactic and diagnosticapplications. Substrates may be targets, for example, when it ispossible to specifically react the bioreactive agent with substrate in amixture such that the reaction creates the conjugate. Such conjugatesare useful when it is desirable to follow a target through a biologicalor other type of system such as when determining the half-life of apharmaceutical.

Photocleavage of conjugates of the invention should preferably notdamage released substrate or impair substrate activity. Proteins,nucleic acids and other protective groups used in peptide and nucleicacid chemistry are known to be stable to most wavelengths of radiationabove 300 nm. PCB carbamates, for example, undergo photolysis uponillumination with long-wave UV light (320-400 nm), resulting in releaseof the unaltered substrate and carbon dioxide (FIG. 7). The yield andexposure time necessary for release of substrate photo-release arestrongly dependent on the structure of photoreactive moiety. In the caseof unsubstituted 2-nitrobenzyl PCB derivatives the yield of photolysisand recovery of the substrate are significantly decreased by theformation of side products which act as internal light filters and arecapable of reacting with amino groups of the substrate. In this case,illumination times vary from about 1 minute to about 24 hours,preferably less than 4 hours, more preferably less than two hours, andeven more preferably less than one hour, and yields are between about 1%to about 95% (V. N. R. Pillai, Synthesis 1, 1980). In the case ofalpha-substituted 2-nitrobenzyl derivatives (methyl, phenyl), there is aconsiderable increase in rate of photo-removal as well as yield of thereleased substrate (J. E. Baldwin et al., Tetrahedron 46:6879, 1990; J.Nargeot et al., Proc. Natl. Acad. Sci. USA 80:2395, 1983).

The choice of a particular bioreactive agent depends on which moleculargroups of the substrate are to be derivatized. For example, reaction ofphotocleavable biotin NHS-ester with a protein results in formation of acovalent bond with primary amino groups such as at the ε-position oflysine residues or the α-NH₂ group at the N-terminal of a protein.Normally, a number of lysine residues are exposed on the surface of aprotein and available for such reaction. Alternatively, several otherphotocleavable biotins can be used which react with hydroxyl groups(—OH) present in tyrosine, threonine and serine residues, carboxylgroups (—COOH) present in aspartate and glutamate residues, andsulfhydryl groups (—SH) present in cysteine residues (Table 4). Thus, awide variety of groups are available which are likely to be on thesurface of a target protein.

Attachment of photocleavable biotin to molecules which bind proteinssuch as receptor ligands, hormones, antibodies, nucleic acids, andproteins that bind glycoproteins can also be accomplished because of thewide variety of reactive groups available for photocleavable biotins.For example, photocleavable biotin can be conveniently linked toantibodies which are directed against a particular protein.Alternatively, photocleavable biotins can be linked to DNA and RNA or toa variety of small molecules including receptor ligands and hormones.The importance of biotinylation of binding-complexes for isolation ofproteins such as membrane receptors and splicesomes has already beendemonstrated using conventional biotins or non-photocleavable biotins.

The choice of the detectable moiety depends on the substrate, itsenvironment and the desired method of detection and isolation. Forexample, a substrate present in low concentrations may require asensitive method of detection such as fluorescent spectroscopy therebyrequiring a fluorescent moiety such as coumarin The wavelength offluorescent emission can be selected by the choice of detectable moietyso as not to interfere with any natural fluorophores which may bepresent in the mixture. In cases where rapid isolation of the substrateis desired, choice of the detectable moiety may be determined by theavailability of a suitable coupling agent. For example, an antigen whichserves as the detectable moiety may be used if a suitable antibody isavailable. Since the detectable moiety, the reactive group and thephotoreactive moieties are chemically separate in the bioreactive agent,the properties of each can be adjusted to meet the multiple requirementsfor detection and isolation of a particular substrate.

Conjugates of the invention may be attached to a solid support via thedetectable moiety, the substrate or any other chemical group of thestructure. The solid support may comprise constructs of glass, ceramic,plastic, metal or a combination of these substances. Useful structuresand constructs include plastic structures such as microtiter plate wellsor the surface of sticks, paddles, beads or microbeads, alloy andinorganic surfaces such as semiconductors, two and three dimensionalhybridization and binding chips, and magnetic beads, chromatographymatrix materials and combinations of these materials. Examples of thechemical structure of conjugates of the invention include:

wherein SUB comprises a substrate; R₁ and R₂ are selected from the groupconsisting of hydrogen, alkyls, substituted alkyls, aryls, substitutedaryls, —CF₃, —NO₂, —COOH and —COOR, and may be the same or different; Ais a divalent functional group selected from the group consisting of—O—, —S— and —NR₁; Y comprises one or more polyatomic groups which maybe the same or different; V comprises one or more optional monoatomicgroups which may be the same or different; Q comprises an optionalspacer moiety; m1 and m2 are integers between 1-5 which can be the sameor different; and D comprises a selectively detectable moiety which isdistinct from R₁ and R₂.

As discussed above, the polyatomic group may be one or more nitrogroups, alkyl groups, alkoxyl groups, or derivatives or combinationsthereof. The optional monoatomic group may be one or more fluoro,chloro, bromo or iodo groups, or hydrogen. The polyatomic and monoatomicgroups and the chemical moieties at R₁ and R₂ may effect thephotocleavage reaction such as the frequency of radiation that willinitiate photocleavage or the the exposure time needed to execute acleavage event. The spacer moiety (Q) may be a branched or unbranchedhydrocarbon or a polymeric carbohydrate and is preferably represented bythe formula:

wherein W and W′ are each selected from the group consisting of —CO—,—CO—NH—, —HN—CO—, —NH—, —O—, —S— and —CH₂—, and may be the same ordifferent; and n1 and n2 are integers from 0-10 which can be the same ordifferent and if either n1 or n2 is zero, then W and W′ are optional.Specific examples of conjugates of the invention are depicted in FIG. 8.

Another embodiment of the invention is directed to conjugates which arepharmaceutical compositions. Compositions must be safe and nontoxic andcan be administered to patients such as humans and other mammals.Composition may be mixed with a pharmaceutically acceptable carrier suchas water, oils, lipids, saccharides, polysaccharides, glycerols,collagens and combinations thereof and administered to patients.

Pharmaceutical compositions with photo-releasable substrates are useful,for example, for delivery of pharmaceutical agents which have shorthalf-lives. Such agents cannot be administered through current meanswithout being subject to inactivation before having an effect.Pharmaceutical agents in the form of conjugates, covalently bound tobioreactive agents, are more stable than isolated agents. After generaladministration of the composition to the patient, the site to be treatedis exposed to appropriate radiation releasing substrate which producesan immediate positive response in a patient. Uncoupling from thebioreactive agent at the point of maximal biological effect is anadvantage unavailable using current administration or stabilizationprocedures. In an analogous fashion, other areas of the patient's bodymay be protected from the biological effect of the pharmaceutical agent.Consequently, using these conjugates, site-directed and site-specificdelivery of a pharmaceutical agent is possible.

Another embodiment of the invention is directed to a method forisolating targets from a heterologous mixture. Bioreactive agents arecontacted with the mixture to react with target forming the conjugate.Alternatively, conjugates can be contacted with the heterologous mixtureto couple substrate within the conjugate to one or more targets.Conjugates can be separated from the mixture by any currently availabletechniques (e.g. Table 5).

TABLE 5 Affinity Techniques Using Avidin Material Method of SeparationMagnetic beads coated with Magnetic separation Streptavidin Beads coatedwith Streptavidin Washing (e.g. centrifugation) and elution BiotinylatedAntibodies Immunoprecipitation Cross-linked-bisacrylamide/azolactoneColumn Chromatography copolymers with avidin Agarose coated withStreptavidin Column Chromatography

Procedures such as chemical or physical separation of components of themixture, electrophoresis, electroelution, sedimentation, centrifugation,filtration, magnetic separation, chemical extraction, affinityseparation methods such as affinity chromatography or anotherchromatographic procedure such as ion-exchange, gradient separation,HPLC or FPLC, and combinations of these techniques are well-known andallow for a rapid isolation with a high efficiency of recovery (e.g. M.Wilchek et al., Methods Enzymol. 184, 1990; M. Wilchek et al., Anal.Biochem 171:1, 1988). After separation or isolation, targets can beeasily quantitated using available methods such as optical absorbance ortransmission (e.g. nucleic acid, proteins, lipids) or the Bradford (M.Bradford, Anal. Biochem. 72:248, 1976) or Lowry (O. Lowry et al., J.Biol. Chem. 193:265, 1951) assays (e.g. proteins), both of which arecommercially available. After separation, coupled conjugates are treatedwith electromagnetic radiation to release substrate. The substratetargets can than be separated from the released bioreactive agent, ifdesired, to obtain substantially or completely pure targets.

Targets which can be detected and isolated in a highly purified form bythis method include nearly any chemical, molecule or macromoleculeincluding immune system modulators, agents of the hematopoietic system,cytokines, proteins, hormones, gene products, antigens, cells includingfetal and stem cells, toxins, bacteria, membrane vesicles, virusparticles, and combinations thereof. Detection and isolation aredetermined by the ability of the bioreactive agent to bind substrate.For example, nucleic acids can be base-paired to complementary nucleicacids, to nucleic acid binding proteins or to chemical moieties whichreact specifically with chemical moieties found on nucleic acids.Proteins can be bound with monoclonal or polyclonal antibodies orantibody fragments specific to those proteins, or chemical moietieswhich react specifically with chemical moieties found on the proteins ofinterest. Substrates may be, for example, precursors of targets such asone or more of the naturally or non-naturally occurring amino acidswherein the target is a nascent protein, or one or more ribonucleotides,deoxyribonucleotide or primers when the target is a nucleic acid.Precursor can be incorporated into target molecules by, for example, invivo or in vitro replication, transcription or translation. Target maybe a protein or protein-containing complex, nucleic acid, gene sequenceor PCR product. Substrates may also be receptors which bind to orotherwise associate with ligands specific for the receptor molecules.Receptors which can be isolated include cytokines wherein the target isa cytokine receptor and antigens wherein the target is an antibody.Preferred conjugates for the detection and isolation of a target from aheterologous mixture are photocleavable biotins linked to antibodies(polyclonal, monoclonal, fragments), photocleavable coumarins linked toantibodies, photocleavable dansyls linked to lipids and derivatives andmodifications thereof.

The heterologous mixture which contains target may be a biologicalsample, any proteinaceous composition such as a cellular or cell-freeextract, nucleic acid containing compositions, a biomass containing, forexample, vegetative or microbial material, a cell culture of primary orimmortalized cells, lipid vesicles or even animals. Animals may be usedto detect targets which may be present in the body or parts of the bodyor, alternatively, to collect and isolate targets such as macromoleculesor cells from animal models. Substrate can also be proteins, peptides,amino acids, amino acid analogs, nucleosides, nucleotides, lipids,vesicles, detergent micells, fatty acids, saccharides, polysaccharides,inorganic molecules, metals and derivatives and combinations thereof

In an application of this method, the substrate may be an integralcomponent of the target such as a nucleotide in the detection andisolation of nascent nucleic acids or an amino acid in the detection andisolation of nascent proteins. Substrate is incorporated into target bychemical or enzymatic techniques and detected and isolated by thepresence of the detectable moiety. Briefly, conjugates are contactedwith reagents in a heterologous mixture such as, for example, in areplication, transcription, translation or coupledtranscription/translation system. Substrates are incorporated intotargets through the action of components in the system such as enzymes,precursor molecules and other reagents of the system. Conjugate coupledtargets are separated from the mixture and treated with electromagneticradiation to release the .target which is then isolated.

Conjugates can be contacted with a heterologous mixture by incubation asin, for example, the enzymatic incorporation of a macromolecularprecursor into a nascent macromolecule which may be either in vivo or invitro. Nucleic acid polymerases will incorporate precursor nucleotidesor nucleic acid primers into nucleic acids. In vitro incubations incell-free reaction mixture are typically performed at a temperature ofbetween about 4° C. to about 45° C., preferably at between about 12° C.to about 37° C., and more preferably at about room temperature.Incubation of conjugates into nascent macromolecules may be complete inabout 5 minutes, about 15 minutes, or about one hour depending on theincubation conditions, or may require two, three or more hours tocomplete. When the heterologous mixture is an animal or an animal modelin vivo incubations are generally performed at body temperature and mayrequire hours or days for conjugates to distribute to areas of theanimal's body which may be remote from the site of introduction, forconjugates to react with targets and for conjugates coupled with targetsto be collected.

One of the preferred embodiments of the invention relates to thedetection or isolation of protein using photocleavable biotin. In oneapplication of this embodiment, PCB is reacted with a protein throughthe formation of covalent bonds with specific chemicals groups of theprotein forming a conjugate. The protein may be either the target to beisolated or detected or a probe for the target protein such as anantibody. The target protein can then be isolated using streptavidinaffinity methodology.

Another application of this embodiment is directed to the use ofphotocleavable biotin to isolate nascent proteins that can be createdfrom in vitro or in vivo protein synthesis. Basically, photocleavablebiotins are synthesized and linked to amino acids (PCB-amino acids)containing special blocking groups. These conjugates are charged to tRNAmolecules and incorporated into peptides and proteins using atranslation or coupled transcription/translation system. PCB-amino acidsof the invention have the property that once illuminated with light, aphotocleavage occurs that produces a native amino acid plus the freebiotin derivative. Such proteins can be isolated in a structurallyand/or functionally unaltered form.

The detailed procedure for the production of photocleavable biotin aminoacids and their incorporation into the nascent proteins involves a fewbasic steps. First, photocleavable biotin is synthesized and linked toan amino acid with an appropriate blocking group. These PCB-amino acidconjugates are charged to tRNA molecules and subsequently incorporatedinto nascent proteins in an in vivo or in vitro translation system.Alternatively, a tRNA molecule is first charged enzymatically with anamino acid such as lysine which is then coupled to a reactive PCB.Nascent proteins are separated and isolated from the other components ofsynthesis using immobilized streptavidin. Photocleavage ofPCB-streptavidin complex from the nascent protein generates a pure andnative, nascent protein.

PCB is attached to an amino acid using, for example, the side-chaingroups such as an amino group (lysine), aliphatic and phenolic hydroxylgroups (serine, threonine and tyrosine), sulfhydryl group (cysteines)and carboxylate group (aspartic and glutamic acids) (FIG. 9). Synthesiscan be achieved by direct condensations with appropriately protectedparent amino acids. For example, lysine side chain amino group can bemodified with PCB by modification of the ε-amino group. The synthesisof, for example, PCB-methionine involves primarily α-amino groupmodification. PCB-methionine can be charged to an initiator tRNA whichcan participate in protein synthesis only at initiation sites whichresults in single PCB incorporation per copy of the nascent protein.

One method for incorporation of a photocleavable biotin amino acid intoa nascent protein involves misaminoacylation of tRNA Normally, a speciesof tRNA is charged by a single, cognate native amino acid. Thisselective charging, termed here enzymatic aminoacylation, isaccomplished by enzymes called aminoacyl-tRNA synthetases and requiresthat the amino acid to be charged to a tRNA molecule be structurallysimilar to a native amino acid. Chemical misaminoacylation can be usedto charge a tRNA with a non-native amino acids such as photocleavableamino acids. The specific steps in chemical misaminoacylation of tRNAsare depicted in FIG. 10.

As shown, tRNA molecules are first truncated to remove the 3′-terminalresidues by successive treatments with periodate, lysine (pH 8.0) andalkaline phosphate (Neu et al., J. Biol. Chem. 239:2927-34, 1964).Alternatively, truncation can be performed by genetic manipulation,whereby a truncated gene coding for the tRNA molecule is constructed andtranscribed to produce truncated tRNA molecules (Sampson et al., Proc.Natl. Acad. Sci. USA 85:1033, 1988). Second, protected acylateddinucleotides, pdCpA, are synthesized (Hudson, J. Org. Chem. 53:617,1988; E. Happ, J. Org. Chem. 52:5387, 1987). PCB-amino acids blockedappropriately at their side chains and/or at α-amino groups, usingstandard protecting groups like Fmoc, are prepared and coupled with thesynthetic dinucleotide in the presence of carboxy group activatingreagents. Subsequent deprotection of Fmoc groups yields aminoacylateddinucleotide.

Third, the photocleavable biotin amino acid is ligated to the truncatedtRNA through the deprotected dinucleotide. The bond formed by thisprocess is different from that resulting from tRNA activation by anaminoacyl-tRNA synthetase, however, the ultimate product is the same. T4RNA ligase does not recognize the O-acyl substituent, and is thusinsensitive to the nature of the attached amino acid (FIG. 10).Misaminoacylation of a variety of non-native amino acids can be easilyperformed. The process is highly sensitive and specific for thestructures of the tRNA and the amino acid.

Aminoacylated tRNA linked to a photocleavable biotin amino acid can alsobe created by employing a conventional aminoacyl synthetase toaminoacylate a tRNA with a native amino acid or by employing specializedchemical reactions which specifically modify the native amino acidlinked to the tRNA to produce a photocleavable biotin aminoacyl-tRNAderivative. These reactions are referred to as post-aminoacylationmodifications. Such post aminoacylation modifications do not fall underthe method of misaminoacylation, since the tRNA is first aminoacylatedwith its cognate described amino acid.

In contrast to chemical aminoacylation, the use of post-aminoacylationmodifications to incorporate photocleavable biotin non-native aminoacids into nascent proteins is very useful since it avoids many of thesteps including in misaminoacylation. Furthermore, many of thephotocleavable biotin derivatives can be prepared which have reactivegroups reacting specifically with desired side chain of amino acids. Forexample, postaminoacylation modification of lysine-tRNA^(Lys), anN-hydroxysuccinimide derivative of PCB can prepared that would reactwith easily accessible primary ε-amino and minimize reactions occurringwith other nucleophilic groups on the tRNA or α-amino groups of theamino acylated native amino acid. These other non-specific modificationscan alter the structure of the tRNA structure and severely compromiseits participation in protein synthesis. Incomplete chain formation couldalso occur when the α-amino group of the amino acid is modified.Post-aminoacylation modifications to incorporate lysine-biotinnon-native amino acids into nascent proteins has been demonstrated(tRNA^(nacend)™; Promega; Madison, Wis.) used for the detection ofnascent protein containing biotin using Western Blots followed byenzymatic assays for biotin (T. V. Kurzchalia et al., Eur. J. Biochem.172:663-68, 1988). However, these biotin derivatives are notphotocleavable which, in the case of NHS-derivatives of PCB, allows thebiotin linkage to the lysine to be photochemically cleaved.

PCB-amino acids can also be incorporated into polypeptide by means ofsolid-support peptide synthesis. First, PCB-amino acids are derivatizedusing base labile fluorenylmethyloxy carbonyl (Fmoc) group for theprotection of α-amino function and acid labile t-butyl derivatives forprotection of reactive side chains. Synthesis is carried out on apolyamide-type resin. Amino acids are activated for coupling assymmetrical anhydrides or pentafluorophenyl esters (E. Atherton et al.,Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989). Second, aminoacids and PCB are coupled and the PCB-amino acid integrated into thepolypeptide chain. Side chain PCB-derivatives, like ε-amino-Lys, sidechain PCB-amino acid esters of Glu and Asp, esters of Ser, Thr and Tyr,are used for incorporation at any site of the polypeptide. PCB-aminoacids may also be incorporated in a site-specific manner into the chainat either predetermined positions or at the N-terminus of the chainusing, for example, PCB-derivatized methionine attached to the initiatortRNA.

A wide range of polypeptides can be formed from PCB-amino acidscytokines and recombinant proteins both eukaryotic and prokaryotic (e.g.α-, β- or γ-interferons; interleukin-1, -2, -3, etc.; epidermal,fibroblastic, stem cell and other types of growth factors), and hormonessuch as the adrenocorticotropic hormones (ACTHs), insulin, theparathyroid hormone (bPTH), the transforming growth factor β (TGF-β) andthe gonadotropin releasing hormone (GnRH) (M. Wilchek et al., MethodsEnzymol. 184:243, 1990; F. M. Finn et al., Methods Enzymol. 184:244,1990; W. Newman et al., Methods Enzymol. 184:275, 1990; E. Hazum,Methods Enzymol. 184:285, 1990). These hormones retain their bindingspecificity for the hormone receptor. One example is the GnRH hormonewhere a biotin was attached to the epsilon amino group Lys-6 throughreaction of a d-biotin p-nitophenyl ester. This biotinylated hormone canbe used for isolation of the GnRH receptor using avidin coated columns.

After incorporation or attachment of PCB into a protein, protein-complexor other amino acid-containing target, the target is isolated using asimple four step procedure (FIG. 11). First, a bioreactive agent (PCB)is synthesized. Second, a substrate is coupled to the bioreactive agentforming a conjugate. Third, target is separated from other materials inthe mixture through the selective interaction of the photocleavablebiotin with avidin, streptavidin or their derivatives. Captured targetsmay be immobilized on a solid support such as magnetic beads, affinitycolumn packing materials or filters which facilitates removal ofcontaminants. Finally, the photocleavable biotin is detached from thetarget by illumination of a wavelength which causes the photocleavablebiotin covalent linkage to be broken. Targets are dissolved or suspendedin solution at a desired concentration. In those situations whereinconjugate coupled targets are not attached to solid supports, release oftargets can be followed by another magnetic capture to remove magneticparticles now containing avidin/streptavidin bound biotin moietyreleased form the photocleavage of PCB. Thus, a completely unalteredprotein is released in any solution chosen, in a purified form and atnearly any concentration desired.

Another example for the use of PCB is where the conjugate comprises aPCB-coupled antibody. The use of photocleavable biotin provides a meansfor recovering target molecule and the antibody for subsequent use.Release of a protein from binding complex can be performed subsequent torelease of the binding complex from the immobilized streptavidin. Thisis an advantage since it enables the release to be performed under wellcontrolled conditions. For example, elution of a target protein from anaffinity column often requires changes in buffer and/or use of acompetitive agent such as an epitope which competes for an antibodybinding site. This can require long exposure of the protein to damagingconditions or the need for increased amounts of the competitive agentwhich can be prohibitively expensive. In contrast, once the proteincomplex is removed from the immobilized streptavidin by photocleavage,the complex can be separated more conveniently. In the case whereantibodies are used as the substrate, an additional advantage of theinvention is that the antibody can also be recovered in an unaltered andpurified form.

A simple scheme for using a PCB-antibody is shown in FIGS. 12A and 12B.Target protein/antibody conjugated to photocleavable biotin is thenimmobilized with streptavidin. Target protein/antibody is decoupled byillumination with light and the target protein/antibody released. Targetprotein is separated from antibody using, for example, increased ordecreased salt (NaCl, KCl) concentrations and recovered. Alternatively,as shown in FIG. 12B, the target is removed from the antibody by usingan epitope-PCB conjugate which competes for the antibody binding site.The antibody-epitope-PCB conjugate is then isolated from the target byimmobilization with streptavidin. The antibody-epitope complex is thenreleased by photocleaving the biotin-epitope complex. A wide variety ofother molecules that interact with proteins can also be utilized inconjunction with PCB for protein isolation, as shown in Table 2,including polypeptides, protein complexes and small ligands.

PCB derivatives attached or incorporated into antibodies or othermacromolecules such as DNA which serve as hybridization probes can alsobe used advantageously for sequential multiple detection of targets. Asin the case of conventional assays for target based onstreptavidin-biotin interactions, the presence of the target is signaledby a streptavidin-enzyme complex which binds to the biotinylated probeand produces an amplified signal by converting a substrate into aproduct which is easily detectable due to a distinctive physicalproperty such as color or luminescence. However, in contrast toconventional biotins, the PCB derivatives can be completely removedallowing for separation removal of the streptavidin-enzyme complex andsubsequent addition of new probes and streptavidin complexes for thedetection of additional target molecules. A similar advantage exists forcytochemical labeling based on streptavidin-biotin interaction. In thiscase, the label can be completely removed by light, allowing foradditional specific cytochemical labels to be used.

In another application of the preferred embodiment, photocleavablebiotin or a PCB derivative can be incorporated into a DNA(deoxyribonucleic acid), RNA (ribonucleic acid) or PNA (polynucleicamide; P. E. Nielsen et al., Sci. 254:1497-1500, 1991) molecule producedby, for example, chemical synthesis, PCR, nick translation or DNA or RNApolymerases (Table 6). Target DNA or RNA can then be isolated by usingstreptavidin affinity methods similar to methods discussed above.Isolation is accomplished, for example, using commercially availablemagnetic beads coated with streptavidin. Beads will bind tightly to allDNA and RNA containing the PCB derivative, whereas all other moleculesare washed away. The photocleavable biotin is then removed from thetarget nucleic acid by illumination.

The incorporation of PCB into nucleic acids such as DNA and RNA involvesthe synthesis and use of compounds that are formed from thederivatization of nucleotides with photocleavable biotins. Examples ofthe nucleotides modified using photocleavable biotin are shown FIG. 13A.

Non-specific incorporation of PCB moieties into nucleic acids isachieved, for example, using bisulfite catalyzed cytosine transaminationand subsequent reaction with PCB-NHS ester or PCB-NH-NH₂(PCB-hydrazide).Alternatively, 5′-phosphate can be converted into phosphoramidite byreaction with water soluble carbodiimide, imidazole and diamine and theresulting phosphoramidite reacted with PCB-NHS ester.

Numerous methods have been developed to introduce an amino group on5′-end of protected oligonucleotides during solid support synthesis.These include carbonyl diimidazole mediated modification of 5′-OH withdiamine and introduction of aliphatic amine moiety on 5′- or 3′-endduring synthesis using special phosphoramidites. Bifunctionalphosphoramidites have also been developed that allow for incorporationof reactive amino group into multiple sites in syntheticoligonucleotides. These methods produce oligonucleotide products bearingaliphatic amino groups, which can be easily converted intoPCB-carbamates by reaction with respective PCB-NHS esters. In ananalogous manner as conventional biotin, PCB moieties can be directlyincorporated into oligonucleotides during solid support synthesis usingrespective PCB-phosphoramidites.

Synthetic oligonucleotides of predetermined or random sequences have avariety of uses as, for example, primers, hybridization probes andantisense sequences. The synthesis of DNA and RNA oligonucleotidesutilizes phosphoramidite chemistry and is routinely performed on anautomated synthesizer with the growing nucleic acid chains attached to asolid support such as CPG (controlled pore glass). In this manner,phosphoramidites and other reagents can be added in excess and removedby filtration The synthesis cycle comprises four reactions. First, acidlabile trityl groups are removed from the 5′-OH groups. Second,phosphoramidites are coupled to the 5′-OH. Third, unreacted 5′-OH groupsare protected by capping with acetyl groups. Finally, internucleotidelinkage is converted from phosphite to phosphotriester by oxidation.This cycle is repeated until the desired sequence is obtained afterwhich, oligonucleotide is cleaved from solid support and purified using,for example, gel electrophoresis and HPLC.

Coupling efficiency for each step is preferably as high as possible suchas greater than 90% or about 97-99%. Unreacted molecules are eliminatedat each step by capping with acetyl groups preventing the formation ofundesired sequences. Crude oligonucleotide contains besides full lengthsequence numerous shorter sequences also called the failure sequences.Purification of such a complex mixture is difficult especially when itcomes to isolation of full-length sequence from slightly shorter failuresequences (e.g. n−1; n−2). This problem becomes even more difficult whensynthesizing long sequences of DNA or RNA where coupling efficienciesare lower and the number of failure sequences higher.5′-PCB-phosphoramidites can be used to selectively labile full-lengtholigonucleotides at their 5′-end during solid-support synthesis onautomated nucleic acid synthesizers.

PCB-phosphoramidites can contain the PCB moiety linked to aphosphoramidite functionality through a spacer arm allowing forefficient binding to avidin. This reagent selectively reacts with the5′-OH group on a sugar ring and can be used on automated synthesizers.Biotinylated sequences can also be prepared using standard synthesiscycles with coupling efficiency being monitored by trityl analysis.Typical coupling efficiencies are between about 90-95%. In addition,photocleavage of 5′-PCB nucleic acid results in formation of5′-phosphorylated sequences. 5′-phosphorylated oligonucleotides arerequired for most applications in molecular biology.

Alternatively, PCB moieties can also be incorporated during synthesisinto oligonucleotides at any position including the 3′-terminus, andinto multiple sites using PCB-phosphoramidites utilizing, for example,bifunctional non-nucleosidic backbones. Enzymes including the Taq DNApolymerase used in PCR reactions, and other DNA and RNA polymerases arecapable of incorporating biotinylated nucleotides into nucleic. acids.PCR technology comprises the process of amplifying one or more specificnucleic acid sequences in a nucleic acid using primers and agents forenzymatic polymerization followed by detection of the now amplifiedsequence (R. K. Saild et al., Sci. 230:1350, 1985; T. J. White et al.,Trends Gent. 5:185, 1989). The basic techniques are described in U.S.Pat. No. 4,683,195, which is specifically incorporated by reference, andvariations thereof described in U.S. Pat. Nos. 5,043,272, 5,057,410 and5,106,727, which are also specifically incorporated by reference.Several examples exist where biotinylated nucleotides have beenefficiently incorporated during the PCR applications. These studiesdemonstrate that PCR carried out in the presence of PCB-nucleotidesresults in a large amplification of target DNA fragment and simultaneouslabeling with PCB. The primers required for PCR reaction may also belabeled.

Table 6 lists different enzymatic methods that would allow incorporationof PCB-nucleotides to generate labeled probes for many applications likein situ hybridization and PCR.

TABLE 6 Enzymatic Methods for Incorporation of PCB-Nucleotides MethodSubstrate Enzyme Remarks Nick Translation DNA or E. coli DNA pol I Mostpopular RNA method Replacement double T4 DNA High specific synthesisusing T4 stranded Polymerase incorporation in DNA polymerase DNA dsDNAReverse RNA Molony murine Preparation of transcriptase leukemia viruslong cDNA copies reverse of RNA transcriptase 3′-Terminal ds DNAterminal 3′-hydroxyl labeling deoxynucleotidyl terminal labelingtransferase of dsDNA RNA labeling RNA SP6 Polymerase T3 RNA is the T3 orT7 RNA most efficient for Polymerase RNA labeling Post-transcription RNASP6, T3 or T7 Pol Incorporate labeling allylamine-UTP first followed byits modification by PCB 3′-labeling of RNA RNA (in- T4 RNA ligase Basedon ADP cluding derivatives of PCB. tRNAs etc)

Biotin-avidin technology is currently used extensively in the field ofmolecular biology and biomedicine as a means for efficiently isolatingthe products of DNA and RNA synthesis as well as for detection ofspecific sequences in nucleic acids. Isolation normally involves theattachment or incorporation of biotin into the DNA or RNA followed byseparation through the interaction of the biotin with streptavidin. Forexample, this methodology is used widely for the isolation of DNA, whichis the product of the polymerase chain reaction. Detection typicallyinvolves the preparation of biotin labeled nucleic acid probes. Theseprobes have found wide-spread application in gene structure and genefunction studies, the diagnosis of human, animal and plant pathogens,and the detection of human genetic abnormalities.

However, conventional methodologies are limited by the difficulty ofdetaching or releasing biotin from the nucleic acid. In particular, itis highly desirable to obtain unaltered DNA or RNA after it is separatedby the biotin-avidin interaction. For example, the presence of biotin onthe nascent DNA can interfere with its subsequent utilization in cloningor hybridization analysis. In addition, the inability to remove biotinfrom a biotinylated nucleic acid probe after an enzyme linked assayprevents additional hybridization assays from being performed on thesame sample.

The utilization of photocleavable biotins in the isolation or detectionof nucleic acids eliminates many of the difficulties listed above byproviding a rapid and effective method of removing the biotin in asingle step. In the case of biotin-avidin based isolation of DNA thisalso accomplishes the step of releasing the DNA from the immobilizedavidin.

In a preferred embodiment of this invention, the isolation of nucleicacids is based on three basic steps. First, a photocleavable biotinderivative is attached to a nucleic acid molecule by enzymatic orchemical means or, alternatively, by incorporation of a photocleavablebiotin nucleotide into a nucleic acid by enzymatic or chemical means.The choice of a particular photocleavable biotin depends on whichmolecular groups are to be derivatized on the nucleic acid. For example,attachment of photocleavable biotin to a nucleic acid can beaccomplished by forming a covalent bond with the aromatic amine, sugarhydroxyls or phosphate groups (Table 4). PCB can also be incorporatedinto oligonucleotides through chemical or enzymatic means. Next, thenucleic acid molecule is separated through the selective interaction ofthe photocleavable biotin with avidin, streptavidin or their derivativeswhich can be immobilized on a material such as magnetic beads, affinitycolumn packing materials or filters. Methods for the separation ofnucleic acids from other molecules in a complex mixture usingphotocleavable biotin are well-established and similar to the moreconventional methods utilizing non-cleavable biotin. This typicallyinvolves an affinity technique based on streptavidin-biotin interactionwhereby the nucleic acid containing biotin is immobilized due to itsinteraction with streptavidin. These techniques include, as shown inTable 5, streptavidin-coated magnetic beads, streptavidin-sepharosecolumns and streptavidin conjugated filters, all of which arecommercially available. For example, nucleic acid molecules containingPCB either through attachment or incorporation are isolated usingstreptavidin-coated magnetic beads. The pool of unbound biomolecules isthen: washed to remove other reactants, buffer and salts. Finally, thephotocleavable biotin is detached from the nucleic acid by illuminationat a wavelength which causes the photocleavable biotin covalent linkageto be broken. The bioreactive agent can be removed leaving asubstantially or completely pure nucleic acid.

Another aspect of the invention is directed to the use of photocleavableconjugates in conjunction with PCR amplification. Methods for theisolation of a PCR product may use one or more oligonucleotide primersas substrates. The nucleic acid sequence of the target is PCR amplifiedusing the conjugated primers. Covalent bonds between the primer and thebioreactive agent are selectively cleavable with electromagneticradiation to release the amplified sequences. Nucleotide sequences whichcan be selectively amplified by this method includes nucleotidesequences found in biological samples, bacterial DNA and eukaryotic DNA.In contrast to conventional biotins, PCB offers an effective method tocompletely remove biotinylated DNA product of the polymerase chainreaction and provides a means to simultaneously release the PCR productfrom the avidin or streptavidin binding medium and remove thebiotinylation in a single step.

Other advantages over conventional biotins include the elimination ofthe need for special reagents or buffers. After photocleavage of biotinfrom the PCR product, the resulting DNA is suitable for cloning andother common usages in molecular biology. After photocleavage of biotinfrom the PCR product, it can be accurately analyzed with standardanalytical methods such as gel electrophoresis. Hybridization probescontaining PCB can be sterilized, wherein the biotin is completelyremoved so that the target DNA can be reprobed using a secondbiotinylated probe. The PCB incorporated into DNA retains the highbinding affinity to avidin unlike the several derivatives of biotinwhere properties of biotin are attenuated for the purposes of easyrelease (e.g. iminobiotin).

Two methods for the isolation of PCR products using PCB are representedin FIGS. 14A and 14B. In both methods, the source of the initial pool ofcells from which the target DNA is to be amplified can be from a widevariety of sources including peripheral blood and biopsy tissues. Aftercell lysis, the crude extract of total genome is subjected to the PCR.

In method A (FIG. 14A), DNA primers are synthesized from the flankingsequences of the target DNA with photocleavable biotin incorporation atthe 5′ ends. For this purpose, a PCB-phosphoramidite can be introduceddirectly at the 5′ end of the oligonucleotide primer during DNAsynthesis. A set of PCR cycles is carried out using normal dNTPs. Thisprocedure results in PCR product where the photocleavable biotin ispresent on the 5′ end of each complementary strand. PCR products areseparated from the mixture containing other components of the PCRmixture including nucleotides, enzymes, buffers by using streptavidinsuch as present on coated magnetic beads (e.g. Dynabeads M-280Streptavidin) which bind the photocleavable biotin present at the 5′ endof the DNA. A typical procedure to be followed is to mix 40 μl of washedDynabeads M-280 Streptavidin with 40 μl of the PCR mixture and toincubate for 15 minutes at room temperature. The Dynabeads are thencollected using magnetic means such as the DYNAL Magnetic ParticleConcentrators. Residual primers will be bound to thestreptavidin-binding material due to the presence of photocleavablebiotin at its 5′ end. A small spin column like NAP-5 (Pharmacia Biotech;Piscataway, N.J.) can be used to remove smaller molecules and primersbefore streptavidin-magnetic bead capture is carried out. Theimmobilized PCR product, now bound to streptavidin, is photolyzed torelease biotin from the DNA and the unmodified PCR product recovered.

In method B (FIG. 14B), the problem of primers contamination iseliminated. DNA primers are synthesized for the flanking sequences ofthe target DNA without 5′ biotinylation using conventionaloligonucleotide synthesis. Normal dNtPs along with a small pool ofPCB-dUTP are introduced into the PCR reactions. For convenience, thedNTPs and PCB-dUTP can be premixed into aliquots to be used inconjunction with PCR reactions. The PCR products are separated from themixture containing other components of the PCR mixture includingnucleotides, enzymes, buffers and primers by using streptavidin such aspresent on coated magnetic beads (e.g. Dynabeads M-280 Streptavidin).

The immobilized PCR product, now bound to streptavidin, is photolyzed torelease biotin from the DNA and recover unmodified PCR product. Method Amay sometimes be preferable when the immobilized DNA which is bound tostreptavidin is to be assayed using a biotinylated probe. In this casethe entire complex could be released after assay by photocleavagefollowed by an addition separation step, eliminating the biotinylatedprobe and leaving the PCR product free for further use such as forcloning. Alternatively, Method B may be preferable if release of aprimer-free product is required without an intermediate assay. Thiswould also produce a higher recovery since there are more PCB moleculesper molecule of DNA.

PCR is also widely used in the detection of a variety of diseasesrelated markers. Table 7 illustrates the various uses of PCR fordetection of diseases and disorders and the potential uses ofPCB-incorporated PCR in such diagnostic and forensic applications.

TABLE 7 PCR use for detection of disease related DNA/RNA Assay of thePCR Disease/Virus/Bacteria Primer product HTLV-I various targets in-Liquid hybridization/Spot cluding tax, gag or env blot HIV targeted atthe con- Oligomer Hybridization served regions of the virus Hepatitis-Bpol gene Southern hybridization Papillomaviruses Dot-blot Restrictionenzyme analysis Cytomegalovirus Hybridization (herpes virus group)Enterovirus 100% conserved RT-PCR followed by regions RNA hybridizationGastroenteritis Reverse hybridization Cholera

In a preferred embodiment of the invention, the methodology, based onPCB, for isolation and detection of PCR products can be applied todiagnostic assays of a variety of diseases, the detection of mutationsas well as to the identification of unique DNA sequences. For example,serological assays such as Western blots and immunofluorescence andradioimmunoprecipitation assays provide a rapid and sensitive procedureto screen for the presence of antibodies to HIV-1. Further, in currentPCR-based analysis, highly conserved regions of the viral genomes aretargeted for amplification and involve hybridization using ³²P-labeledoligomer probes in solution to one strand of an amplified product. Thesetests can be used only for the direct detection of the virus. A usefulassay for the detection of HIV would detect not only active virus, butalso the presence of latent virus which has not yet expressed itsgenome, but is still present in cells. This would allow determination ofboth latent and actively replicating virus. This would be particularlyuseful in newborns where maternal antibodies can interfere withserological tests.

In another preferred embodiment, conjugates may be used to efficientlycreate genomic or cDNA libraries. Construction of a PCR-directed cDNAlibrary from total RNA may provide the only methodological approach toanalyze cell-specific gene expression where the amount of biologicaltissue is severely restricted. This approach is particularly applicablewhere a specific stimulus results in the modification or differentiationof a small number of cells within a population.

Current schemes for the construction of, for example, cDNA librariesrequire the isolation of cellular RNA and usually further purificationof mRNA from the more abundant rRNA and tRNA components. The majoradvantage in PCR based methods is that mRNA purification is notnecessary. This is particularly advantageous where biological materialis limited wherein efficient mRNA purification would be impossible. Thegeneral strategy is illustrated schematically in FIG. 15.

High quality, intact RNA is prepared using guanidinium hydrochlorideprocedure (S. J. Gurr et al., PCR: A Practical Approach, OxfordUniversity Press, New York, 1991). First strand cDNA is synthesizedusing AMV reverse transcriptase in the presence of PCB-dCTP in 1:1 ratiowith dCIP (final concentration of both should equal that of otherdNTPs). This is followed by removal of oligo-dT primers which can becarried out in a rapid and quantitative manner using magnetic capturefollowed by illumination. Current methods use CTAB precipitation whichresults in loss of valuable cDNA:RNA hybrids, which is followed byhomopolymer tailing using oligo-dG (A. Otsuka, Gene 13:339, 1981). Afterhomopolymer tailing, the RNA is hydrolyzed which subjects cDNA to harshcondition such as 50 mM NaOH at 65° C. These steps are not necessaryusing PCB-nucleotide conjugates. Second strand synthesis and cDNAamplification is achieved by PCR in presence of PCB-nucleotides. All thePCR products are ethanol precipitated and are ligated with a suitablydigested vector. These vectors are rapidly purified by using magneticcapture and illumination. These vectors can be screened usingPCB-modified hybridization probes to selectively remove the vector ofinterest. This vector can then be magnetically captured and illuminatedto obtain pure vector containing the DNA of interest.

Another aspect of the invention facilitates the process of site-directedmutagenesis. For example, cassette mutagenesis is a powerful approach increating site directed mutants and avoids sequencing of entire genes toconfirm the introduction of a mutation. Basic steps in cassettemutagenesis require construction of a vector which contains the gene ofinterest with well-separated, unique restriction sites. Restrictiondigestion of vector carrying the gene of interest using two uniquerestriction enzymes to remove a cassette of double-stranded DNA wherethe mutation is to be introduced.

The cassette containing the desired mutation is synthesized usingautomated oligonucleotide synthesis. The digested vector and thecassette are ligated to generate complete vector. These ligated mixturesare transformed into host cells and the colonies are screened bysequencing the cassette regions of plasmid mini-preps. Although thisprocess is capable of rapidly and accurately generating a large numberof site directed mutants as shown in case of bacteriorhodopsin (H. G.Khorana, J. Biol. Chem. 263:7439, 1988), there are several areas wheretime and resources can be saved using PCB.

After the initial vector restriction, the new mutant containing cassetteis labeled either chemically or enzymatically with PCB. Subsequentligation mixture is purified using streptavidin interaction, forexample, magnetic capture using Dynabead-280 streptavidin. The captureresults in selective isolation of recombinant vector containing thePCB-cassette and free PCB-labeled cassette. Photolysis releases themutant containing vector in pure form in any desired solution andconcentration. Subsequent transformants have very high probability ofcontaining only the desired mutant.

In current protocols, after restriction digestion of vector, completepurification of doubly restricted vector by, for example, agarose gelelectrophoresis, is often difficult. The size difference of theresulting DNA fragments is typically very small. For example, the sizeof a typical cassette is about 30 base-pairs (bp) and a typical vectorabout 5000 bp. Restriction enzyme digestion to remove the cassette wouldproduce fragments of 5000 bp and 30 bp which can be readilydistinguished and isolated. However, this is assuming that completedigestion has occurred. Partial digestion would produce an additionalfragment of 5030 bp which is not easily detected much less distinguishedor isolated. Thus, ineffective purification of restricted vector createsa higher chance of ligation without incorporation of the mutantcassette. Use of PCB avoids gel electrophoresis and subsequent elutionof restricted fragments. Magnetic capture can be performed even in theligation mixture.

Liposomes are widely used for targeting and introduction of biologicallyimportant materials into cells via fusion into the cell membrane (G.Gregoriadis editor, Liposome Technology, vol. III, CRC Press, BocaRaton, Fla., 1984). The avidin-biotin system is useful for in vitrostudies to mediate between encapsulated liposomes and target cells.These studies involve the use of biotin containing phospholipids tointroduce biotin into membranes (E. A. Bayer et al., Biochim. Biophys.Acta 550:464, 1979). Avidin-biotin system has also been attempted fortargeting drugs into cells. These studies have determined that theeffects of biotinylation on the properties of the modified lipid andbiotinylated molecule, albeit chemically altered, maintains theirfundamental properties. For example, the biotinylated lipid is fullyextractable in organic solvents, forms liposomes and mixed liposomes,and is correctly oriented in the latter (B. Rivnay et al., MethodsEnzymol. 149:119, 1987). Fatty acid components comprise the interior ofthe bilayer and the biotinyl head groups are exposed to the aqueousenvironment of the solvent. Thus, the use of biotinylated lipids forliposome preparation allows an almost irreversible binding ofbiotin-streptavidin interaction. Although biotin and its interactionallows easy manipulation of these liposomes, any attempt to concentrateor separate these liposomes using immobilized streptavidin, such asmagnetic beads coated with streptavidin, fails as it is virtuallyimpossible to separate the concentrated/separated liposomes from boundstreptavidin.

Use of PCB-lipids readily overcomes these limitations as illuminationreleases the PCB-lipid containing liposomes into desired solutions atdesired concentrations. The steps involved in the manipulations ofliposomes using PCB-lipids include (1) attachment of a photocleavablebiotin derivative to a liposome by chemical means or, alternatively,incorporation of a photocleavable biotin lipid (PCB-lipid) into aliposome by first derivatizing the lipid with PCB followed bypreparation of liposome (PCB-liposome), (2) concentration or separationof the PCB-liposome through the selective interaction of thephotocleavable biotin with avidin, streptavidin or their derivativeswhich is normally immobilized on a material such as magnetic beads,affinity column packing materials and filters, and (3) detachment of thephotocleavable biotin from the PCB-liposome by illumination at awavelength which causes the photocleavable biotin covalent linkage to bebroken

The methods for attachment of various photocleavable biotins directly toliposomes involves modification of the functional groups on the lipidsmolecules using PCB. The choice of a particular photocleavable biotindepends on which molecular groups are to be derivatized on the lipidsconstituting the liposome. For example, attachment of photocleavablebiotin to a liposome could be accomplished by forming a covalent bondwith the amino group on the phosphatidylserine. Although a number ofgroup-specific PCB derivatives are available that allow modification ofany functional group to yield PCB-lipid, phosphatidylethanolamine andphosphatidylserine are exemplary. PCB-phosphatidylserine andPCB-phosphatidylethanolamine are shown in FIG. 16.

Concentration and separation of liposome from a heterologous mixture isreadily achieved using photocleavable biotin by established proceduresthat are similar to the more conventional methods utilizingnon-cleavable biotin lipids. This normally involves an affinitytechnique based on streptavidin-biotin interaction whereby the liposomescontaining biotin are immobilized due to their interaction withstreptavidin. These techniques include streptavidin-coated magneticbeads, streptavidin-sepharose columns and streptavidin conjugatedfilters, all of which are commercially available. After concentratingPCB-liposomes using affinity interaction with streptavidin, liposomescan be released by illumination in desired solution and at the desiredconcentration.

Avidin-biotin technology is used extensively in the field of affinitycytochemistry where specific cell structures or subcellular componentsare localized by selective labeling. Two different approaches termedimmunohistochemistry (IHC) and in situ hybridization (ISH) areavailable. In IHC, a primary antibody or binding ligand which isbiotinylated (alternatively, a biotinylated secondary antibody can beutilized), is directed at a specific antigen on the surface of a cell orcellular structure. The cell or cellular structure is then localized byapplication of a reporter complex which could consist of anavidin-enzyme conjugate, avidin-fluorescence marker or avidin-ferritincomplex for electron microscopic localization.

In ISH, a DNA probe which is biotinylated is used to label the locationof specific mRNA or DNA sequence in individual cells or tissue sections.A similar range of avidin based reporter complexes can be used as inimmunohistochemistry including enzyme, fluorescence and ferritinconjugated avidins. However, a serious limitation of the conventionalapplication of these two techniques is the difficulty of removing thebiotin-marker complex once a label has been applied to sample. Removalof the label would enable additional labels to be applied therebyproviding a means to map the interrelationship between various cellularand subcellular components in a tissue. The use of PCB provides aconvenient means to achieve this goal since photocleavage of the linkerconnecting the antibody or DNA probe and PCB will result in release ofthe marker complex.

In situ hybridization techniques are used to detect specific cellularDNA or which are non-uniformly distributed in individual cells or tissuesections, and to detect viral nucleic acid sequences which are oftenfocal in distribution. In contrast to Southern-, Northern- or dot-blothybridization assays which determine the average content of targetmolecules per cell in the extracted tissue, ISH detects specific targetsequences that are focally distributed in a small number of cells thatcontain significant levels of target molecules (E. J. Gowans et al.,Nucleic Acid Probes, R. H. Symons editor, CRC Press, Boca Raton, Fla.,1989).

Current technology is limited by fact that a single tissue section,chromosome slide or cell-slide is usable only once and probing thedistribution of second target which could be another set of sequencesthat are co-regulated or correlated, is almost impossible. PCB labeledprobes offer completely non-invasive approach for multiple in situhybridizations on such valuable sample. Combined with multiple in situhybridizations, a composite picture can be constructed of distributionof various nucleic acids. The protocol for ISH is adapted from publishedprocedures (D J. Brigati et al., Virol. 126:32, 1983; I.Guerin-Reverchon et al., J. Immunol. Meth. 123:167, 1989). The procedureinvolves careful fixation and sectioning of tissues which may be eitherparaffin or frozen sections. Fixation is designed not only to fixnucleic acids, but also to bind the section firmly to the slide.Glutaraldehyde is used for DNA and paraformaldehyde is used for RNAdetection. Both steps include optional denaturation steps which arenecessary if dsDNA or dsRNA is the target of reaction. Further stepsinvolve preparation of different probes that are labeled using PCB, andhybridization of these probes in a sequential manner. ISH follows thesame general principles as a solution and filter hybridization (R. J.Britton et al., Nucleic Acid Hybridization, B. D. Hames and S. J.Higgins editors, IRL Press, Oxford, 1985). PCB-labeled probe is thendetected using variety techniques that use streptavidin conjugateddetecting systems. A picture is obtained that shows distribution offirst probe in the tissue section or chromosome picture. Photolysisresults in release of detection assembly along with biotin moiety. ISHand detection is then repeated with second probe and its distribution isobtained. A composite picture is created that shows distribution of avariety of probes in the tissue section.

In situ hybridization (ISH) techniques are also used to detect eitherspecific cellular DNA or RNA sequences at the chromosomal level inindividual cells or tissue sections. The method is referred to ashybridization histochemistry. ISH is ideally suited not only to thedetection of cellular nucleic acid sequences which are non-uniformlydistributed in tissues or cell, but also to the detection of viralnucleic acid sequences which are often focal in distribution Thedetection of nucleic acid by ISH satisfies the primary objective ofreflecting accurately the intercellular and intracellular distributionof target molecules in the sample. In general, ISH may be used to detectDNA corresponding to normal or abnormal genes, to identify thechromosomal location of particular DNA sequences, and to measure thelevel of expression of these genes by mRNA detection

In particular, mRNA is a common target for in situ hybridizationreaction in studies of gene expression and cell differentiation In thespecial cases of virus infected cells, either viral genomic nucleic acidor RNA transcripts can be detected. ISH is especially valuable where thehistological mapping of target cells within a tissue is sought. Incontrast, Southern, Northern and dot-blot hybridization assays measurethe overall concentration of the target molecules per cell in theextracted tissue. If PCB is substituted for biotin in the application ofimmunochemistry and in situ hybridization, the methodology forlocalization of a label is almost identical. However, an importantadvantage is the ability to completely remove the label in the form of aPCB-avidin marker complex by simple illumination of the sample whichphotocleaves the PCB linkage to the antibody or hybridization probe.This step renders the sample, typically a tissue section, available forfurther sequential labeling by additional different probes.

There is often a need to determine if antigenic peptides, hormones orviral gene products detected intracellularly by immunohistochemicaltechniques represent de novo synthesis or merely deposition and passiveaccumulation It is also useful to determine if specific mRNA, detectedby ISH is translated into protein product. The use of PCB facilitatessuch a determination since sequential interrogation of a single sampleis possible using the same enzyme-avidin linked reporter complex. Thisapproach avoids complications due to the use of different samples.

Many different genes, difficult or impossible to locate otherwise, canbe localized using conjugates of the invention. Neurotransmitterreceptors are members of large gene families. By a combination ofexpression strategies and homology cloning, dozens of receptor genes inthis family have now been cloned. These genes include those encodingreceptors for glutamate, glycine, and γ-aminobutyric acid (GABA)receptors. Cloning has revealed the existence of distinctneurotransmitter-receptors in numbers that had not been anticipated byneurophysiologists. The significance of this surprising receptorhomogeneity is not yet known but ISH has shown that individual receptorsubtypes are expressed in unique patterns in the brain. An importantgoal is to determine the distribution of these receptors in the brain.Ordinarily, this is done using many thin slices each exposed to adifferent hybridization probe. However, these experiments suffer fromthe use of multiple tissue sections. In contrast, the use of PCB wouldallow repeated analysis of the same rat brain section to determinecomplete distribution of each receptor expression.

Further, cloned genes and markers can be localized by ISH to specificregions of chromosomes. Sequences can be localized to sub-chromosomalregions by hybridizing radioactively labeled probes directly tochromosome spreads. Chromosome spreads are made by using cells whosedivision has been blocked in the metaphase by a chemical like colcemidwhich disrupts the mitotic spindle. After fixing and staining, a patternof light and dark bands develops on each chromosome, so that thechromosomes can be identified. After ISH, location of the radioactiveprobe is revealed by the distribution of silver grains in a photographicemulsion layered over the spread. However, the detection of singlecopies of human genes is difficult and can be done only by pooling thedistribution of grains over as many as 30 or more metaphase spreads.

PCB offers the unique advantage that the probe can be photoreleasedafter localization of a specific genetic sequence on a chromophore and asecond probe applied. In cases where too closely spaced sequences are tobe detected, prior removal of the probe may be essential in order toavoid interference from the original probe. In addition, the methodallows for the use of a large number of probes to be appliedsequentially and is not limited by the availability of different markercomplexes which can be simultaneously measured. This technique couldalso be used advantageously for rapid ordering of multiple probes on achromosome.

An important application of biotin-avidin technology is the separationof cells from a complex mixture often containing a variety of differentcell types. Cells which can be utilized include cells within abiological sample, tissue culture cells, bacterial cells and diseasedcells. Cells may be mammalian, such as mammalian stem or fetal cells.Receptors include antibodies directed against the classical cell surfacereceptors and other surface proteins, but also any cell-surface moleculethat has a specific affinity for another molecule. Preferably, thereceptor is an antibody which recognizes a cell surface marker on thetarget cell or a specific protein which recognizes a cell surface ligandor other macromolecule on the target cell. PCB-modified antibodies,specific for surface antigens, receptors or ligands on the cell, can beused for cell separation or cell sorting with, for example, an apparatussuch as a fluorescence-activated cell sorter (FACS). ThesePCB-antibodies, when linked to streptavidin-coated magnetic beads canbind to the specific cell population bearing a particular antigen andcan then be separated using ImmunoMagnetic Separation (IMS).

Current methodologies do not allow gentle separation of these magneticparticles from separated or sorted cell population. PCB modifiedantibodies, however, can be readily separated from magnetic particleafter illumination. IMS involving PCB-antibodies can be used incell-sorting, tissue typing and for selective enrichment ofmicroorganisms using modifications of protocols described earlier (A.Elbe, J. Immunol. 149:1694, 1992; S. Qin, Sci. 259:974, 1993; L.Leclerecq, Immunol. Lett. 28:135, 1991).

In conventional methods, a biotinylated antibody is utilized which bindsselectively to an antigen residing only on the target cell. The targetcells can then be isolated from the mixture by using streptavidin-coatedmagnetic beads or streptavidin based affinity columns. The affinitymaterial sometimes contains a secondary antibody directed toward theprimary antibody. A severe limitation of this approach is the difficultyof releasing the cells once they are bound to the affinity mediumthrough the biotin-streptavidin interaction. Normal methods that aredesigned to disrupt the biotin-streptavidin interaction or theantibody-antigen interaction can reduce the overall viability of thereleased cells. This is a serious disadvantage if the cells are to beused later for culturing or transplantation. Similarly, conditions suchas low pH that disrupt the antibody-antigen interaction can cause celldamage. A standard technique is overnight incubation in a culture mediumfollowed by vigorous mixing. This causes shedding of the antigeninvolved in binding. However, this method is time consuming, can lead tocell degradation and does not result in complete release. An alternatemethod is to disrupt the antibody-antigen interaction with enzymatictreatment, which can also be damaging to a cell. An additional method isthe utilization of anti-FAB antibodies to compete for the antigen. Suchmethods are time consuming, expensive due to the use of antibodies andonly partially effective. These methods of detachment (release) of cellsare all particularly ineffective when the antibody-antigen interactionis strong or the binding involves several antigen-antibody interactionsmixing. Conventional biotins which can be chemically cleaved such asNHS-SS-Biotin (sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate; Pierce Chemical; Rockford, Ill.) poseserious problems since the cleavage medium consisting of a highconcentration of reducing agents such as thiols will cause reduction ofprotein disulfide bonds and a subsequent loss of cell viability.

In contrast to conventional methods, photocleavable biotin offers aninexpensive, effective and rapid means to release immobilized cellssimply by using light exposure. Release occurs due to photocleavage ofthe covalent linkage between the antibody and biotin. While the antibodystill remains bound on the released cell, this is normally not a problemfor cell integrity or additional utilization of the separated cells.Since photocleavage can be performed in short periods and results inalmost full removal of the photocleavable biotin release is rapid andcomplete.

Further, using photocleavable conjugates, cells which represent a verysmall populations of sample of cells can be accurately and efficientlyselected. This enables methods such as the selection of immune cells,stem cells, fetal cells, precursor cells and nearly and cell type fromlymph (e.g. interstitial, lymphatic), blood (e.g. arterial andperipheral blood) or tissue (organ, soft tissues, muscle) samples.Selected and isolated cells can then be cultured in very large numbersand possible reintroduced into the same or another patient. Culturedcells can also be used in gene therapy by the introduction of geneticmaterial into cultured cells. Such techniques are not possible usingconventional detection and isolation procedures.

The same approach can also be used by linking photocleavable biotin toother cell specific macromolecules such as cell-associated ligands orantigens. For example, B cells expressing a specific immunoglobulinreceptor for a target antigen could be isolated by attachingphotocleavable biotin to the target antigen and then binding it to astreptavidin-coated bead. Alternatively hybridoma screening and stemcell selection could be performed using this approach.

The three basic steps involved in cell isolation using PCB are (1)attachment of a photocleavable biotin, or molecule containing aphotocleavable biotin derivative including (antibodies, receptorligands, antigen) to the surface of the target cell by a photochemicallycleavable bond, (2) separation of said cell type from other cells andmaterials in the complex mixture through the selective interaction ofthe photocleavable biotin with avidin, streptavidin or theirderivatives, and (3) detachment of the photocleavable biotin from thesaid cell type by illumination at a wavelength which causes thephotocleavable biotin covalent linkage to be broken. These steps, usingPCB, can be carried out in combination with ordinary biotin. In thiscase, cells containing two or more surface markers can be from thosecontaining only one. Each of these steps is illustrated in FIG. 17.

Several applications for the use of PCB for cell separation offers clearadvantages over existing technology. A common method for isolation ofcells is to label the cell with a fluorescent dye which is directed tothe target cell through an antibody-fluorescent conjugate. The cells canthan be separated by using a fluorescence-activated cell sorting deviceand used for a variety of purposes including cell typing, hybridomaproduction and tissue culturing. However, with this technique theseparated cells remain labeled with the fluorescent dye. This can resultin reduction in cell viability, prevent their use in therapeuticapplications where the dye can be toxic, and prevent furtherfluorescence based sorting into subspecies of cells. One example wouldbe the separation of lymphocytes using characteristic antigens such asCD2, CD4, CD8 and CD19 followed by sorting into subspecies.

An alternative approach is to utilize an antibody which is conjugated toPCB. In this case the cell can be fluorescence labeled with anavidin-fluorescein complex which is commercially available in a varietyof forms (Table 8). The fluorescent label can then be removed byphotocleavage of the PCB which results in release of thePCB-avidin-fluorescent complex. A variety of fluorescent labels existwhich absorb in a region outside of the absorption band of the PCB, thusavoiding photocleavage during the cell sorting.

TABLE 8 Commercially Available Avidin-Florescent Dye Complexes ConjugateAbsorption (nm) Emission Producer Avidin- 490 520 Pierce FluoresceinAvidin-R- 450-470 574 Pierce Phycoerythrin Avidin- 515-520 575 PierceRhodamine Avidin- 575 600 Pierce Rhodamine 600 Avidin-Texas 595 615Pierce Red

Another example of the utility of PCB methodology for cell isolation isthe isolation of B cells for hybridoma production Briefly, formation ofhybridomas for the purpose of monoclonal antibody production involvesthe fusion of myeloma cells with lymphocyte B cells. Generally, aheterogeneous population of B cells is utilized which contains differentimmunoglobulin receptors. The hybridoma which expresses the desiredantibody is then screened by assaying for binding to a particularantigen. This screening process can be time-consuming and expensive.Screening could be avoided if a method existed for isolation of aparticular population of B cells which only expressed the desiredimmunoglobulin receptor. In principle, this could be accomplished byusing a biotinylated antigen, such as a polypeptide with a specificsequence, which will only bind to those B cells which express thespecific immunoglobulin receptors for the antigen. This subpopulation ofB cells could then be selected by using avidin affinity techniques suchavidin-coated magnetic beads. However, the subsequent step of hybridomaformation will still be prevented unless the B cells can be releasedfrom the avidin-biotin complex in a viable form.

The use of PCB-biotin avoids this problem by providing a simple methodfor releasing the B cells in a viable form after immobilization by thebiotin-avidin interaction. Photocleavage of the photocleavable biotinlinkage with the antibody results in the release of the B-cells and thebound antigen which is now in an unmodified form Since no chemicaltreatment is required, the cells will retain their viability for furtherfusion to the myeloma cells. Furthermore, the method is rapid andsuitable for automation.

Another embodiment of the invention is directed to targets isolated bythe above method which may be utilized in pharmaceutical compositions totreat or prevent diseases and disorders. Pharmaceutical compositions maycomprise the isolated targets plus a pharmaceutically acceptable carriersuch as water, oils, lipids, saccharides, polysaccharides, glycerols,collagens or combinations of these components. The composition isadministered to patients for the treatment or prevention of certaindiseases and disorders and for the site-directed administration ofpharmaceutical agents.

Another embodiment of the invention is directed to a method fordetermining an in vivo half-life of a pharmaceutical in a patient.Conjugates are formed by coupling the pharmaceutical to a bioreactiveagent via a covalent bond that can be selectively cleaved withelectromagnetic radiation. Conjugates are administered to the patientafter which, two or more biological samples are removed. Samples aretreated with electromagnetic radiation to release the pharmaceuticalfrom the bioreactive agent, the amount of the bioreactive agent in thebiological samples is determined, and the in vivo half-life of thepharmaceutical determined.

The pharmaceutical may be a composition comprising cytokines, immunesystem modulators, agents of the hematopoietic system, chemotherapeuticagents, radio-isotopes, antigens, anti-neoplastic agents, recombinantproteins, enzymes, PCR products, nucleic acids, hormones, vaccines,haptens, toxins, antibiotics, nascent proteins, synthetic andrecombinant pharmaceuticals, and derivatives and combinations of thesecomponents. Conjugates may be administered to patients by parenteraladministration, sublingual administration, enteral administration,pulmonary absorption, topical application and combinations thereof.Animals which can be tested include mammals such as humans, cattle,pigs, sheep, dogs, cats, horses and rodents. Biological samples whichare collected can be sample of peripheral blood, blood plasma, serum,cerebrospinal fluid, lymph, urine, stool, ophthalmic fluids, organs andbodily tissues.

Another embodiment of the invention is directed to the controlledrelease of a substrate into a medium. Conjugates comprised of abioreactive agent coupled to the substrate by a covalent bond which canbe selectively cleaved with electromagnetic radiation are created asdescribed. These conjugates are bound to a surface of an article andplaced into the medium The surface of the article is exposed to ameasured amount of electromagnetic radiation and the substrate releasedinto the medium to carry out a beneficial effect. Alternatively, anarticle may be placed at a selected site and the conjugates, having anaffinity for the article, are administered at a distal site. Conjugatesthen migrate to the selected site and perform an intended function.After completion of that function, radiation is applied and thesubstrate is released from the fixed bioreactive agents. Releasedsubstrate may be naturally eliminated from the patient's system. Thiscan be highly useful, for example, in radiation therapy for cancerpatients.

Preferred are radiation wavelengths which can penetrate the medium.Depending on the amount and frequency of radiation exposure, release canbe controlled and continued over a period of time. This method is usefulfor the controlled and site-directed administration of pharmaceuticalcompositions to a patient. In such cases, the medium in which thearticle is placed may be blood, lymph, interstitial fluid or a tissue.Controlled release may also be performed in tissue culture foradministering a constant or periodic amount of a substrate to a cellculture fluid or balanced salt solution for uptake by the cells.Articles which may be coupled with substrate and placed within oradjacent to a patient's body include articles comprising carbohydrates,lipids, proteins, polysaccharides, cellulose, metals including magnets,organic polymers and combinations thereof. Preferably, the surface ofthe article is coated with streptavidin and the bioreactive agent isphotocleavable biotin.

Alternatively, articles containing conjugates or agents can be placedinto the site of the disorder, such as a tumor. The pharmaceutical agentsuch as, for example, a radioactive agent is administered to the patientand becomes bound to the fixed conjugates or agents. Effectsattributable to the pharmaceutical agent are localized. The article isexposed to a measured amount of electromagnetic radiation and thepharmaceutical agents released into the body and excreted. This methodis preferred when only a short term exposure of the pharmaceutical agentis desired or to efficiently remove potentially harmful agents afterthey have had their desired effects.

Another embodiment of the invention is directed to a method for creatinga photocleavable oligonucleotide. A bioreactive agent is createdcomprised of a photoreactive moiety coupled to a detectable moiety andcontaining a phosphoramidite. The oligonucleotide is synthesized usingconventional phosphoramidite chemistry. The nucleotide precursorscomprise one or more photocleavable phosphoramidites such aspurine-phosphoramidites (uracil, cytosine, thymine) orpyrimidine-phosphoramidites (adenine, guanine) as the ribose ordeoxyribose forms, or derivatives thereof This method can be performedmanually or automated using a commercially available oligonucleotidesynthesizer. Photocleavable oligonucleotides can be utilized as primersor probes, in diagnostic kits and in every instance in which a nucleicacid can be used.

Another embodiment of the invention is directed to a method fordetecting a target molecule in a heterologous mixture. Conjugates areformed by coupling a substrate to a bioreactive agent with a covalentbond that is selectively cleavable with electromagnetic radiationConjugates are contacted with the heterologous mixture to couplesubstrate to one or more target molecules. Uncoupled conjugates areremoved and the coupled conjugate are treated with electromagneticradiation to release the detectable moiety. Presence of target moleculescan be detected by detecting the presence of the released detectablemoiety. Target macromolecules may be proteins, peptides, nucleic acids,lipids, polysaccharides, metallic compounds, virus, bacteria, eukaryoticcells, parasites and derivatives and combinations thereof. Oncedetected, target macromolecules can be isolated and the amount isolatedquantitated by current any of the techniques available to those ofordinary skill in the art.

A specific application of streptavidin-biotin technology is in thedetection of targets in medical diagnosis. Generally, the target is abiotinylated molecule or a biotinylated probe for the target molecule.The interaction of streptavidin with the biotinylated target or probe isamplified by an enzyme conjugated to streptavidin which catalyzes achromogenic reaction. For example, a variety of enzymes conjugated tostreptavidin are commercially available including horseradishperoxidase, β-galactosidase, glucose oxidase and alkaline phosphate.Each of these enzymes catalyze a chromogenic reaction. Additionalmethods of detection include conjugating streptavidin to fluorescent,chemi-fluorescent, radioactive or electron dense molecules.

An example of biotin-avidin interaction in medical diagnosis also formsthe basis for a wide array of enzyme-linked immunospecific assays(ELISA). In this case, an antibody specific for the target molecule, theprimary antibody, or a secondary antibody directed against the primaryantibody is biotinylated. Detection is accomplished with astreptavidin-enzyme conjugate as described. A large number ofimmunoassays have been developed based on this approach. However,multiple immunoassays on the same sample are not easily accomplishedusing conventional technology since there exists no simple method ofremoving the avidin-enzyme-complex once bound to a biotin derivativewithout damaging the sample. In contrast to traditional methodologies,PCB can be reused many times for multiple assays of the same sample formultiple screening of pathogens and other markers of human diseases.

The biotin-avidin interaction also forms the basis of sensitive methodsfor detecting specific nucleic acid sequences such as screening humanDNA samples. DNA or RNA probes which hybridize to a target DNA sequenceare biotinylated. DNA containing the target sequence is detected with anstreptavidin-enzyme conjugate. This method is used in conjunction withthe polymerase chain reaction where the target gene or sequence to bescreened is first amplified using specific primers. The introduction ofbiotin nucleotides into the primer or directly into the nascent PCRproduct using biotinylated nucleotides facilities isolation of thetarget DNA. A variety of biotinylated nucleotides such as biotin-dUTPare available for such purposes. However, this method has at least twolimitations.

First, the presence of biotin in the amplified target DNA prevents theuse of biotinylated probes without prior removal of the biotinylation.The presence of endogenous biotin or biotin-containing molecules in thesample also lowers the sensitivity of this assay. Second, the presenceof biotin in the target DNA lowers hybridization efficiency and hencethe sensitivity of the assay. As discussed above, the use of PCBderivatives such as PCB-nucleotides effectively eliminates theseproblems by allowing for complete removal of biotin from the procedure.

Conjugates and methods of the invention can be used in conjunction witha variety of diagnostic assay involving nascent protein detection. Forexample, diagnostic assays for cancer have been developed which rely onin vitro expression of PCR amplified genes followed by examination ofthe nascent protein product using gel electrophoresis (S. M. Powell etal., N. Engl. J. Med. 329:1982, 1993). The isolation of such proteinsand the subsequent sensitivity of such tests could be increased by theincorporation of PCB amino acids.

Biotin-streptavidin technology is widely used as the basis fornon-radioactive ELISA including diagnostic assays for specificindicators of diseases and disorders such as disease-linked antigensincluding adenovirus antigen (K Mortensson-Egnund et al., J. Virol.Methods 14:57, 1986), bovine leukemia virus (E. N. Esteban et al.,Cancer Res. 45:3231, 1985), flavivirus (E. A. Gould et al., J. Virol.Methods 11:41, 1985), Hepatitis B surface antigen (C. Kendall et al., J.Immol. Methods 56:329, 1983), Herpes simplex virus antigen (K.Adler-Strorthz et al., J. Clin. Microbiol. 18:1329, 1983), respiratorysyncytial virus (A. Hornsleth et al., J. Med. Virol. 18:113, 1986),bacterial antigens (R. H. Yolken et al., J. Immunol. Methods 56:319,1983) and melanoma-associated antigens (human) (AC. Morgan et al.,Cancer Res. 43:3155, 1983). The usefulness of these assays can becompromised if endogenous biotin is present in the sample. In this case,a false background will be obtained since the streptavidin-reporterenzyme complex will react both to non-specific biotins and to thebiotinylated antibodies directed against the target protein. Whileseveral approaches to eliminate background due to non-specific bindingof the avidin or streptavidin to non-biotinylated targets including theuse of high ionic-strength. buffers (C. J. P. Jones et al., Histochem.J. 19:264, 1987), milk proteins (R. C. Duhamel et al., J. Histochem.Cytochem. 33:711, 1985) and lysozyme (E. A. Bayer et al., Anal. Biochem.163:204, 1987) and altered streptavidins such as ImmunoPure NeutrAvidin(Pierce Chemical; Rockford, Ill.), none has been very effective ineliminating background due to endogenous biotin.

Use of photocleavable biotins in conjunction with immunoassaysalleviates the problem by providing a means of determining thebackground level of non-specific biotin. The ELISA assay is firstperformed using conventional methodology except that the antibodydirected against the protein is targeted with photocleavable biotin(FIG. 19). The streptavidin-reporter enzyme complex linked to the probeantibody system is then removed via light cleavage of the photocleavablebiotin. Cleavage does not release the reporter enzyme-avidin complexbound from non-specific bound biotin since this biotin is non-cleavable.The signal obtained from the remaining biotin can be used as a measureof the endogenous biotin present in the sample and subtracted from theprimary signal obtained in step 1.

Background signal due to endogenous biotin in a biotin-avidin basedELISA can be simply detected using photocleavable biotin. The ELISA isperformed according to normal procedures using streptavidin-reporterenzyme complex. However, the streptavidin-reporter enzyme complex isthen removed with light and the system reassayed to determine thebackground level of endogenous biotin.

A second application of photocleavable biotins is its use to conductmultiple biotin based ELISA assays on the same sample. This. is based onthe ability to fully remove the streptavidin-reporter enzyme complexwith light upon photocleavage of the PCB linkage. In this case a secondELISA can-be performed using a different antibody probe and reporterenzyme complex without interference from the first ELISA as shown inFIG. 20. Normally such a multiple ELISA is not possible because thesignal obtained from a second antibody probe combined with astreptavidin-reporter enzyme complex cannot be easily separated from theoriginal signal.

For example, in a conventional biotin-avidin ELISA, thestreptavidin-reporter enzyme complex remains bound to the probe antibodyeven after the chromogenic product of the reporter enzyme is removed.Thus, it will continue to produce a chromogenic product even after asecond ELISA is performed and interfere with the signal of that secondimmunoassay. In contrast, the removal of the streptavidin-enzyme complexby cleavage of the photocleavable biotin with light and then subsequentwashes eliminates this problem since the original reporter enzyme is nolonger present.

In another application of the invention, detection of pathogens such asmicroorganisms from biological material often requires their isolationand culturing. The more effective the isolation step, the more reliableand rapid the culturing step will be because of the elimination of othercontaminants and the concentration of the target pathogen. While avariety of affinity techniques exists for separation of microorganismssuch as magnetic beads conjugated to selective antibodies, the problemof release of the microorganisms in a viable form suitable for culturingand sensitive detection still remains. In contrast, PCB which is linkedto the antibody or binding ligand provides a non-damaging and rapidmeans for photochemical release of the microorganism in a viable form.

For example, this application of the invention provides the basis fordevelopment of rapid diagnostic assays for a variety of pathogensinvolved in human and animal disease that were previously not possibleusing conventional biotin-streptavidin technology. Microorganisms couldalso be isolated from food, milk, soil and other materials for thepurpose of depletion or detection using this approach.

Another embodiment of the invention is directed to methods for treatinga disorder by the controlled release of a therapeutic agent at aselected site. Conjugates are formed by binding a bioreactive agent to atherapeutic agent with a bond that is selectively cleavable withelectromagnetic radiation, wherein the bioreactive agent is comprised ofa directable moiety bonded to a photoreactive moiety and the directablemoiety has an affinity for the selected site. Conjugates areadministered to a patient having the disorder and the selected site issubjected to a measured amount of electromagnetic radiation for thecontrolled release of the therapeutic agent to treat the disorder.Disorders which can be detected include infections, such as bacterialinfections, viral infections and parasitic infections, neoplasias suchas a tumor, and genetic disorders such as an overproduction ordeficiency of an enzyme or other genetic product.

The therapeutic agents may be toxins, immune system modulators,hematopoietic agents, proteins, nucleic acids, substrate analogstranscription and translation factors, antigens and combinationsthereof. Directable moieties may be antibodies such as a monoclonal orpolyclonal antibody or antibody fragment.

Another embodiment of the invention is directed to diagnostic kits fordetecting or screening for diseases and disorders in patients. Kitscontain a conjugate comprised of a bioreactive agent, covalently bondedto a diagnostic agent having an affinity for an indicator of saiddisorder in a biological sample obtained from the patient. The indicatormay be a presence or absence or an increased or decreased amount orlevel of a characteristic marker of the disorder such as an antigen orantibody, a cytokine, a specific cell type (e.g. B cells; cytotoxic,suppressor or helper T cells; macrophages; stem cells), a particularenzyme, nucleic acid or protein. Disorders which can be detected includeinfections, neoplasias and genetic disorders. Infections which can bedetected include bacterial infections, viral infections and parasiticinfections. Neoplasias which can be detected include tumors. Geneticdisorders which can be detected include an overproduction or deficiencyof an enzyme. Biological samples which can be added to the sampleinclude samples of peripheral blood, blood plasma, serum, cerebrospinalfluid, lymph, urine, stool, ophthalmic fluids, organs and bodilytissues. Such kits may also be used to detect or screen for the presenceof fetal or stem cells in a biological sample which can be isolated andcultured or further analyzed.

Kits may also be used to detect the presence of multiple nucleic acidsand/or proteins on, for example, an electroblot using a series ofsecondary probes linked to biotin. After each probe is introduced, thebiotin attachment could be cleaved allowing the enzymatic assay complexto be removed thus providing for a new secondary probe to be introduced.Such an approach would be extremely useful as the basis of medicaldiagnostic assays, where multiple antigens or nucleic acid sequencesneeded to be probed rapidly and automatically.

The kit may also be a nucleic acid mutagenesis kit for use in molecularbiological applications such as introducing or correcting mutations inDNA or RNA. The nucleic acid may be an oligonucleotide for use in PCR orcassette-type applications. Such oligonucleotides may be single-strandedor double-stranded and preferably contain one or more restriction enzymerecognition sequences internally and ligatable 5′ and 3′ ends which alsocontain part of a restriction enzyme recognition site. Alternatively,one or more ends may be blocked to facilitate directed coupling.

The following examples are offered to illustrate various embodiments ofthe invention, but should not be viewed as limiting the scope of theinvention.

EXAMPLES Example 1 Synthesis of Photocleavable Agents

Five grams or 27.6 mmol of 5-methyl-2-nitrobenzoic acid (FIG. 5,compound “6”; Aldrich Chemical; Milwaukee, Wis.) was added in smallportions to 10 ml (16.4 g or 148 mmol) of thionyl chloride withstirring. The mixture was stirred at room temperature for 10 hours.Excess of thionyl chloride was removed by vacuum to give the acidchloride (“7”). Magnesium turnings (1.07 g or 44.2 mmol), absoluteethanol (6 ml), chlorobenzene (8 ml), and 0.1 ml of dry CCl₄, wererefluxed until most of the magnesium reacted. A solution of diethylmalonate (4.82 g or mmol) in 10 ml of chlorobenzene was added followedby the addition of the acid chloride (5.49 g) in 10 ml of chlorobenzene.The reaction mixture stirred for 1 hour and 1.7 ml of concentrated H₂SO₄in 17 ml of H₂O was added, stirred for additional 15 minutes. 20 ml ofchloroform was added and the layers separated. The aqueous layer wasextracted three times with 10 ml and the extracts were combined, driedand evaporated to dryness. Residue was dissolved in 8.25 mls of aceticacid. 5.4 ml of H₂O and 1 ml of concentrated H₂SO₄ were added, themixture was refluxed for 6 hours, neutralized with aqueous Na₂CO₃ andextracted three times with 20 ml of CHCl₃. Extracts were combined, driedand solvents removed by vacuum. Residue was crystallized from 70%ethanol to produce 4.46 g, or about 81%, of5-methyl-2-nitroacetophenone. 5-methyl-2-nitroacetophenone (3.51 g or19.6 mmol), N-bromosuccinimide (3.66 g or 20.6 mmol), and benzoylperoxide (46 mg or 0.01 eq) were refluxed in 20 ml of CCl, for 5 hours.The reaction mixture was filtered, the filtrate concentrated andcrystallized from CCl₄ to produce 3.64 g (72%) of5-bromomethyl-2-nitroacetophenone (“8”). Compound 8 (2.0 g or 7.75 mmol)was added to a solution of hexamethylenetetramine (1.14 g or 8.13 mmol)in 15 ml of chlorobenzene. The mixture was stirred overnight, theprecipitate filtered off and washed with 10 mls of chlorobenzene and 20mls of diethyl ether. The precipitate (2.93 g or 736 mmol) was suspendedin 35 ml of 95% ethanol followed by the addition of concentrated HCl(3.12 ml or 5 eq.). The mixture was stirred overnight and evaporated todryness. Ten mls of DMF were added to the residue followed by theaddition of a 6-biotinamidocaproic acid (3.29 g or 1.25 eq.) in 35 ml ofN-methylpyrrolidone, dicyclohexylcarbodiimide (228 g or 1.5 eq.), andtriethylamine (1.28 ml or 1.25 eq.). The solution was stirred overnightat room temperature, the precipitate filtered off, and filtrateprecipitated to 700 ml of diethyl ether. The precipitate was dried andpurified on a silica gel column using step gradient (5-20%) of MeOH inCHCl₃ to produce 2.27 g (about 58%) of compound 11.

Compound 11 (1 g or 1.87 mmol) was dissolved in 15 ml of 70% EtOH (FIG.5). The solution was cooled to 0° C. and sodium borohydride (141 mg or 4eq.) added. The solution was stirred at 0° C. for 30 minutes and at roomtemperature for an additional 2 hours. The reaction was quenched withthe addition of 1 ml acetone, neutralized with 0.1N HCl, concentrated,the supernatant discarded, the residue washed with water and dried toproduce 0.71 g (about 71%) of compound 12.

Compound 12 (1.07 g or 2 mmol) was dissolved in 10 ml DMF.N,N′-disuccinimidyl carbonate (Fluka Chemical; Ronkonkoma, N.Y.) (1 g,1.5 eq.) was added followed by 0.081 ml or 3 eq. of triethylamine (FIG.5). After 5 hours at room temperature, solvents were evaporated todryness and the residue was washed consecutively with 0.1N NaHCO₃,water, dioxane, diethyl ether and dried to give 1.04 g (about 69%) of5-(5-biotinamidocaproamidomethyl)-1-(2-nitro)phenylethyl-N-hydroxysuccinimidyl carbonate (PCB-NHS) ester (compound13). M.P.=113-114° C. (uncorrected); CI-MS (M⁺=676.5); UV-VISλ=204 nm,ε1=19190 M⁻¹ cm⁻¹; λ2=272 nm, ε2=6350 M⁻¹ cm⁻¹ in phosphate buffer,pH=7.4. ¹H NMR (DMSO-d₆, Varian XL-400 MHz), [δppm]: 8.48 (t, 1H),8.05-8.03 (d, 1H), 7.75-7.71 (t, 1H), 7.66 (s, 1H), 7.46-7.45 (d, 1H),6.44 (s, 1H), 6.37 (s, 1H), 6.28-6.27 (m, 1H), 439 (m, 2H), 4.30 (r,1H), 4.12 (m, 1H), 357 (d, 2H), 3.09 (m, 1H), 3.01-2.99 (m, 2H), 2.79(m, 5H), 2.58-255 (d, 1H), 2.17-2.15 (m, 2H), 2.04-2.02 (m, 2H),1.72-1.71 (m, 2H), 1.66-1.43 (m, br, 6H), 138-136 (m, br, 2H), 1.26-1.25(m, br, 3H); IR (KBr); Vc=o 1815 and 1790 cm⁻¹.

Synthesis of PCB-NHS Ester (FIG. 18):

2-Bromo-2′-nitroacetophenone (“14”) (Aldrich Chemical; Milwaukee, Wis.)(1 g; 4.09 mmol) was converted into 2-amino-2′-nitroacetophenonehydrochloride (“15”) by reaction with 1.05 eq. of hexamethylenetetramineand hydrolysis, and was coupled to 5-biotinamidocaproic acid (1.25 eq.)using DCC (1.5 eq.) in DMF to produce2-(5-biotinamidocaproamido)-2′-nitroaceptophenone (“16”) (about 52%yield) which was reduced (about 75% yield; “17” and converted intoreactive NHS derivative (“18”)2-(5-biotinamidocaproamido)-2′-nitrophenylethyl-N-hydroxysuccinimidylcarbonate (about 69% yield) as described.

Synthesis of PCB-NHS Ester (FIG. 19):

3-amino-4-methoxybenzoic acid (“19”) (Aldrich Chemical; Milwaukee, Wis.)(5 g or 29.9 mmol) was suspended in 40 ml acetic acid. Acetic anhydride(3 ml or 1:04 eq) was added by stirring. The reaction mixture wasstirred for 2 hours at room temperature. 25 ml of 0.1N HCl was added andthe precipitate was filtered off and washed with 3×10 ml of 0.1N HCl and5×10 ml water to produce 5.97 g (about 95%). 3-(N-acetyl)amino-4-methoxybenzoic acid (“20”) (5 g or 23.5 mmol) was added to 20 mlof fuming nitric acid at 0° C. on vigorous stirring. The solution wasstirred at 0° C. for an additional hour and poured onto 200 g of ice.Precipitate was filtered off, washed with 5×20 mls of water and dried toproduce 438 g (about 72%) of 2-nitro-4-methoxy-5 (N-acetyl)aminobenzoicacid (“21”), which was converted into2-nitro-4-methoxy-5-(N-acetyl)aminoacetophenone (“22”) (about 63% yield)as described. 2-nitro-4-methoxy-5-(N-acetyl)aminoacetophenone (“22”) (1g or 4,76 mmol) was dissolved in 5 ml of DMF and the solution was addedto 5-biotinamidocaproyl chloride prepared separately from5-biotinamidocaproic acid (1.55 g or 1 eq.) and 5 ml thionyl chloride.The reaction mixture was stirred overnight at room temperature and addedto 100 ml of diethyl ether. Precipitate was purified using small silicagel column and a step gradient of MeOH in CHCl₃ to give 1.16 g (about47%) of 2-nitro-4-methoxy-5-(5-biotinamidocaproamido) acetophenone(“23”). This intermediate was converted into its corresponding alcohol(about 85% yield) and into the reactive NHS ester derivative5-(5-biotinamidocaproamido)4methoxy-1-(2-nitro)phenylethyl-N-hydroxysuccinimidylcarbonate (“25”) with about a 69% yield.

Synthesis of photocleavable coumarin (FIG. 21):5-aminomethyl-2-nitroacetophenone hydrochloride (“26”) (1.15 g or 5mmol) was dissolved in 20 ml DMF. To this solution7-Methoxycoumarin-4-acetic acid (“27”) (Aldrich Chemical; Milwaukee,Wis.) (1.46 g or 1.25 eq.) and dicyclohexylcarbodiimide (1.55 g or 1.5eq.) was added followed by triethylamine (0.7 ml or 1 eq.). The solutionwas stirred overnight at room temperature, 20 ml of CHCl₃ was added, thesolution was washed with 0.1N NaHCO₃ (3×15 ml), and the organic layerwas dried and purified on a silica gel column using step gradient ofMeOH in CH₂Cl₂ to give 1.37 g (about 64%) of (“28”). Compound 28 (1 g or2.33 mmol) was dissolved in 15 ml of 95% EtOH, the solution cooled to 0°C. and sodium borohydride (176 mg or 4 eq.) added. The solution wasstirred at 0° C. for 1 hour and the reaction was quenched by addition of1 ml acetone and neutralized with 0.1N HCl. The solution was thenextracted with 3×15 ml of CHCl₃. Extracts were combined, dried, andpurified on a silica gel column using step gradient of MeOH in CH₂Cl₂ togive 832 mg (about 83%) of compound 29. Compound 29 (1.29 g or 3 mmol)was dissolved in 10 ml of DMF-acetonitrile (1:1). N,N′-disuccinimidylcarbonate (Fluka Chemical; Ronkonkoma, N.Y.) (1.15 g or 15 eq.) wasadded which was followed with chloroform. The solution was washed with3×15 ml of 0.1N NaHCO₃, solvents were evaporated to dryness and theresidue recrystallized from acetonitrile to give 1.22 g (about 71%) ofphotocleavable coumarin NHS ester (“30”).

Example 2 Synthesis of Photocleavable Conjugates—PCB-Amino Acids

PCB-amino acids were prepared by derivatization of the α-amino group ofthe amino acid. Derivatives were prepared from either PCB chloroformatesor their corresponding N-hydroxysuccinimidyl esters and amino acids in aweakly alkaline media using a modification of the procedure by Sigler etal. (Biopolymers 22:2157, 1983; L. Lapatsanis et al., Can. J. Chem.60:976, 1982; A Paquet, Can. J. Chem. 60:2711, 1977). This procedure wasalso used for the synthesis of ε-NH₂-PCB-Lys wherein the α-amino groupof Lys is protected with Fmoc. PCB-amino acids were also prepared bycarboxyl or hydroxy group derivatization. Briefly, carboxyl residues ofaspartic acid and glutamic acid were esterified using PCB-OH. α-aminogroups were protected as Fmoc derivatives and α-carboxyl groups wereprotected as t-butyl esters. Esterification of the carboxyl side chainwas mediated using dicyclohexylcarbodiimide (DCC) (P. Sieber, Helv. ChimActa 60:2711, 1977). Esterification was also carried out by reactingPCB-chloroformate with the hydroxyl side chains of appropriatelyprotected threonine or serine residues (M. Bednarek et al., Int. J.Pept. Prot. Res. 21:196, 1983; H. Kessler et al., Tetrahedron 281,1983).

Preparation and photocleavage of PCB-Leucine-Enkephaline:Leucine-enkephaline (Sigma Chemical; St. Louis, Mo.) (15.5 μmol/ml in0.1N NaHCO₃, pH 8.0) and PCB-NHS ester (17 μmol/ml in DMF) were mixedand stirred overnight at room temperature. At this time HPLC analysisshowed complete conversion of Leu-Enk into PCB-Leu-Enk. HPLC analysiswas performed on a Waters System (Waters Chromatography; Marlboro,Mass.) comprising of U6K injector, 600 Controller, Novapak C₁₀-Column(3.9×150 mm) and 996 Photodiode Array detector. Tests were performedusing a linear gradient 30-45% of B over 10 minutes followed by 45% of Bisocratic for 10 minutes.

PCB-Leu Enk (1.93 μmol/ml in phosphate buffer, pH 7.4) was irradiatedwith a long-wavelength, UV-lamp (Blak Ray XX-15 UV lamp; UVP Inc; SanGabriel, Calif.) at a distance of 15 cm (emission peak 365 nm, lampintensity=1.1 mW/cm² at a distance of 31 cm). HPLC analysis showedcomplete photorelease of Leu-Enk within 5 minutes and confirmedauthenticity of the released material on the basis of retention time andUV spectra.

PCB-Leu-Enk (10 nmoles) was incubated for 30 minutes in a suspension ofmonomeric-avidin agarose beads (15 nmoles). The suspension wasspun-filtered for 3 minutes (16,000×). Binding efficiency was determinedat about 94%. Sample was resuspended in phosphate buffer (2 ml) andirradiated as described. Released Leu-Enk was assayed usingfluorescamine. Emission spectra were measured on a SLM 48000 fluorimeterusing 380 nm excitation (λ=488 nm). Photorelease of Leu-Enk wasquantitated after 5 minutes of illumination.

Example 3 Solid Phase Synthesis of PCB-Polypeptides

PCB-amino acids were incorporated into polypeptides by solid-supportpeptide synthesis. Standard method for employing base labilefluorenylmethyloxy (Fmoc) group for the protection of α-amino functionand acid labile t-butyl derivatives for protection of α-carboxyl andreactive side chains were used. Synthesis was carried out on apolyamide-type resin. Amino acids were activated for coupling assymmetrical anhydrides or pentafluorophenyl esters (E. Atherton et al.,Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989). The PCB-aminoacid for site-specific incorporation into polypeptide chain wasderivatized with PCB moiety. Side chain PCB-derivatives, likeε-amino-Lys, side chain PCB-AA esters of Glu and Asp, and esters of Ser,Thr and Tyr, were incorporated within the polypeptide. These PCB-aminoacids were stable during solid phase peptide synthesis, in 20%piperidine/DMF (Fmoc removal) and 1-95% trifluoroacetic acid (t-Bu,t-Boc removal, cleavage of the peptide from polyamide resin) (E.Atherton et al., Solid Phase Peptide Synthesis, IRL Press, Oxford,1989).

Example 4 Detection and Isolation of Nascent Proteins

Misaminoacylation of tRNA: The general strategy used for generatingmisaminoacylated tRNA is shown in FIG. 10 and involves truncation oftRNA molecules, dinucleotide synthesis, aminoacylation of thedinucleotide and ligase mediated coupling.

Truncated tRNA molecules were generated by periodate degradation in thepresence of lysine and alkaline phosphatase basically as described byNeu and Heppel (J. Biol. Chem. 239:2927-34, 1964). Briefly, 4 mmoles ofuncharged E. coli tRNA^(Lys) molecules (Sigma Chemical; St. Louis, Mo.)were truncated with two successive treatments of 50 mM sodiummetaperiodate and 0.5 M lysine, pH 9.0, at 60° C. for 30 minutes in atotal volume of 50 μl. Reaction conditions were always above 50° C. andutilized a 10-fold excess of metaperiodate. Excess periodate wasdestroyed treatment with 5 μl of 1M glycerol. The pH of the solution wasadjusted to 8.5 by adding 15 μl of Tris-HCl to a final concentration of0.1 M. The reaction volume was increased to 150 μl by adding 100 μl ofwater. Alkaline phosphatase (15 μl, 30 units) was added and the reactionmixture incubated again at 60° C. for two hours. Incubation was followedby ethanol precipitation of total tRNA, ethanol washing, drying thepellet and dissolving the pellet in 20 μl water. This process wasrepeated twice to obtain the truncated tRNA.

Dinucleotide synthesis was carried out basically as performed by Hudson(J. Org. Chem. 53:617-24, 1988), and can be described as a three stepprocess, deoxycytidine protection, adenosine protection and dinucleotidesynthesis.

Deoxycytidine protection: All reaction were conducted at roomtemperature unless otherwise indicated. First, the 5′ and 3′ hydroxylgroups of deoxycytidine were protected by reacting with 4.1 equivalentsof trimethylsilyl chloride for 2 hours with constant stirring. Exocyclicamine function was protected by reacting it with 1.1 equivalents ofFmoc-Cl for 3 hours. Deprotection of the 5′ and 3′ hydroxyl wasaccomplished by the addition of 0.05 equivalents of KF and incubationfor 30 minutes. The resulting product was produced at an 87% yield.Phosphate groups were added by incubating this compound with 1equivalent of bis-(2-chlorophenyl) phosphorochloridate and incubatingthe mixture for 2 hours at 0° C. The yield in this case was 25-30%.

Adenosine protection: Trimethylsilyl chloride (4.1 equivalents) wasadded to adenosine residue and incubated for 2 hours, after which, 1.1equivalents of Fmoc-Cl introduced and incubation continued for 3 hours.The TMS groups were deprotected with 0.5 equivalents of fluoride ions asdescribed above. The Fmoc protected adenosine was obtained in a 56%yield. To further protect the 2′-hydroxyl, compound 22 was reacted with1.1 equivalents of tetraisopropyl disiloxyl chloride (TIPDSCl₂) for 3hours which produces compound 23 at a 68-70% yield. The compound wasconverted to compound 24 by incubation with 20 equivalents ofdihydropyran and 0.33 equivalents of p-toluenesulfonic acid in dioxanefor about 4-5 hours. This compound was directly converted withoutisolation by the addition of 8 equivalents of tetrabutyl ammoniumfluoride in a mixture of tetrahydro-furan, pyridine and water.

Dinucleotide synthesis: The protected deoxycytidine, compound 20 (FIG.19), and the protected adenosine were coupled by the addition of 1.1equivalents of 2-chlorophenyl bis-(1-hydroxy benzotriazolyl) phosphatein tetrahydrofuran with constant stirring for 30 minutes. This wasfollowed by the addition of 13 equivalents of protected adenosine in thepresence of N-methylimidazole for 30 minutes. The coupling yield wasabout 70% and the proton NMR spectrum of the coupled product is asfollows: (δ 8.76 m, 2H), (δ 8.0 m, 3H), (δ 7.8 m, 3H) (δ 7.6 m, 4H), (δ7.5 m, 3H), (δ 7.4 m, 18H), (δ 7.0 m, 2H), (δ 4.85 m, 14H), (δ 4.25 m,1H); (δ 3.6 m, 2H), (δ 3.2 m, 2H) (δ 2.9 m, 3H), (δ 2.6 m, 1H), (δ2.0-1.2 m, 7H).

Aminoacylation of the dinucleotide was accomplished by linking theNα-protected Nε-PCB-lys, to the dinucleotide with an ester linkage.First, the protected amino acid was activated with 6 equivalents ofbenzotriazol-1-yl-oxy tris-(dimethylamino) phosphonium hexafluorophosphate and 60 equivalents of 1-hydroxybenzotriazole intetrahydrofuran. The mixture was incubated for 20 minutes withcontinuous stirring. This was followed with the addition of 1 equivalentof dinucleotide in 3 equivalents N-methylimidazole, and the reactioncontinued at room temperature for 2 hours. Deprotection was carried outby the addition of tetramethyl guanidine and 4-nitrobenzaldoxime, andcontinuous stirring for another 3 hours. The reaction was completed bythe addition of acetic acid and incubation, again with continuousstirring for 30 minutes at 0° C. which produced the aminoacylateddinucleotide.

Ligation of the tRNA to the aminoacylated dinucleotide was performedbasically as described by T. G. Heckler et al. (Tetrahedron 40: 87-94,1984). Briefly, truncated tRNA molecules (8.6 O.D.₂₆₀ units/ml) andaminoacylated dinucleotides (4.6 O.D.₂₆₀ units/ml), were incubated with340 units/ml T4 RNA ligase for 16 hours at 4° C. The reaction bufferincluded 55 mM Na-Hepes, pH 7.5, 15 MM MgCl₂, 250 μM ATP, 20 μg/ml BSAand 10% DMSO. After incubation, the reaction mixture was diluted to afinal concentration of 50 mM NaOAc, pH 45, containing 10 mM MgCl₂. Theresulting mixture was applied to a DEAE-cellulose column (1 ml),equilibrated with 50 mM NaOAc, pH 45, 10 mM MgCl₂, at 4° C. The columnwas washed with 0.25 mM NaCl to remove RNA ligase and other non-tRNAcomponents. The tRNA-containing factions were pooled and loaded onto aBD-cellulose column at 4° C., that had been equilibrated with 50 mMNaOAc, pH 4.5, 10 mM MgCl₂, and 1.0 M NaCl. Unreacted tRNA was removedby washes with 10 ml of the same buffer. Pure misaminoacylated tRNA wasobtained by eluting the column with buffer containing 25% ethanol.

Preparation of extract: Wheat germ embryo extract was prepared byfloatation of wheat germs to enrich for embryos using a mixture ofcyclohexane and carbon tetrachloride (1:6), followed by drying overnight(about 14 hours). Floated wheat germ embryos (5 g) were ground in amortar with 5 grams of powdered glass to obtain a fine powder.Extraction medium (Buffer I: 10 mM tris-acetate buffer, pH 7.6, 1 nMmagnesium acetate, 90 mM potassium acetate, and 1 mM DTT) was added tosmall portions until a smooth paste was obtained. The homogenatecontaining disrupted embryos and 25 ml of extraction medium wascentrifuged twice at 23,000×g. The extract was applied to a sephadexG-25 fine column and eluted in Buffer II (10 mM tris-acetate buffer, pH7.6, 3 mM magnesium acetate, 50 mM potassium acetate, and 1 mM DTT). Abright yellow band migrating in void volume and was collected (S-23) asone ml fractions which were frozen in liquid nitrogen.

Preparation of template: Template DNA was prepared by linearizingplasmid pSP72-bop with EcoRI. Restricted linear template DNA waspurified by phenol extraction and DNA precipitation. Large scale mRNAsynthesis was carried out by in vitro transcription using theSP6-ribomax system (Promega; Madison, Wis.). Purified mRNA was denaturedat 67° C. for 10 minutes immediately prior to use.

Cell-Free Translation Reactions: The incorporation mixture (100 μl)contained 50 μl of S-23 extract, 5 mM magnesium acetate, 5 mMtris-acetate, pH 7.6, 20 mM Hepes-KOH buffer, pH 7.5; 100 mM potassiumacetate, 0.5 mM DTT, 0.375 mM GTP, 2.5 mM ATP, 10 mM creatine phosphate,60 μg/ml creatine kinase, and 100 μg/ml mRNA containing the geneticsequence which codes for bacteriorhodopsin. Misaminoacylated PCB-lysinewas added at 170 μg/ml and concentrations of magnesium ions and ATP wereoptimized. The mixture was incubated at 25° C. for one hour.

Isolation of Nascent Proteins Containing PCB-Lysine: Streptavidin coatedmagnetic Dynabeads M-280 (Dynal; Oslo, Norway), having a bindingcapacity of 10 μg of biotinylated protein per mg of bead. Beads atconcentrations of 2 mg/mL were washed at least 3 times to removestabilizing BSA. The translation mixture containing PCB-lysineincorporated into nascent protein was mixed with streptavidin coatedbeads and incubated at room temperature for 30 minutes. A magnetic fieldwas applied using a magnetic particle concentrator (MPC) (Dynal; Oslo,Norway) for 0.5-1.0 minute and the supernatant removed with pipettes.The reaction mixture was washed 3 times and the magnetic beads suspendedin 50 μof water.

Photolysis was carried out in a quartz cuvette using a Black-Ray longwave UV lamp, Model B-100 (UV Products, Inc.; San Gabriel, Calif.). Theemission peak intensity was approximately 1100 μW/cm² at 365 nm.Magnetic capture was repeated to remove the beads. Nascent proteinsobtained were quantitated and yields estimated at 70-95%.

Example 5 In Vitro Synthesis of Nascent Proteins using PhotocleavableConjugates

Post-Aminoacylation Linkage: A schematic representation of the stepsinvolved in incorporation of PCB-amino acid for the detection and/orisolation of targets using post-aminoacylation linkage is shown in FIG.10. E. coli tRNA^(Lys) (Sigma Chem.; St. Louis, Mo.) was aminoacylatedwith lysine (A. E. Johnson et al., Proc. Natl. Acad. Sci. USA 75:3075,1978). The NHS ester of PCB (compound 13) dissolved in dimethylsulphoxide, was added at 0° C. to the solution of Lys-tRNA^(Lys) and themodified tRNA purified using benzoylated DEAE-cellulose column (U. C.Kreig et al., Proc. Natl. Acad. Sci. USA 83:8604, 1986). mRNA wastranslated in a cell-free, wheat-germ system as described by Sonar etal. (Biochem. 32:13777, 1993). Nascent proteins containing PCB-lysinewere purified by acetone precipitation to remove PCB-lysyl tRNA followedby magnetic capture of nascent proteins containing PCB-lysine usingstreptavidin coated magnetic beads. Material obtained after magneticcapture was irradiated for 10 minutes to release nascent protein.

Example 6 Synthesis of Photocleavable Conjugates—PCB Nucleotides

Synthesis of PCB-dUTP (FIG. 13A): 5-(3-Aminoallyl)-dUTP ammonium salt(“31”) (Sigma Chemical; St. Louis, Mo.) (10 mg or 16.6 μmol) wasdissolved in 200 μl of 0.1N NaHCO₃. To this solution was added asolution of PCB-NHS (compound 13; 12.5 mg or 1 eq.) in 100 μl of DMF.The reaction mixture was stirred overnight at room temperature,concentrated, and purified by reverse-phase semi-preparative HPLC(Novapak C₁₈ column; Waters Chromatography; Marlboro, Mass.) using a10-50% linear gradient of acetonitrile (B) in 5 mM triethylammoniumacetate (A) over 30 minutes. Fractions containing PCB-dUTP were pooled,lyophilized, and redissolved in TE buffer (pH 7.4) to a concentration of5 mM, and the solution used for enzymatic incorporation into nucleicacids (yield about 56%). Similar procedures were used to preparePCB-UTP, PCB-(d)ATP, and PCB-(d)CTP, using5-(3-aminohexyl)-(deoxy)cytidine triphosphate, respectively.

Example 7 Synthesis of Photocleavable Conjugates—PCB Phosphoramidites

Synthesis of PCB-phosphoramidite (FIG. 13B):5-(-5-biotinamidocapro-amidomethyl)-2-nitroacetophenone (“37”) (534 mgor 1 mmol) was made anhydrous by coevaporation with pyridine (3×2 ml)and dissolved in 5 ml of pyridine. 4,4′-dimethoxytrityl chloride (406 mgor 1.2 eq) and 4-dimethylaminopyridine (6 mg or 0.05 eq) were added andthe resulting solution stirred at room temperature for 24 hours. Ten mlof CHCl₃ and 20 ml of 0.1N aqueous NaHCO₃ were added, the layers formedseparated and the organic layer dried, evaporated to dryness andpurified on a silica gel column using 0-5% step gradient of MeOH inCHCl₃ to give 576 mg (about 69%) of compound 38. Intermediate 38 (836 mgor 1 mmol) was dissolved in 8 ml of 95% EtOH. The solution was cooled to0° C. and vigorously stirred. To the solution was added NaBH₄ (19 mg or2 eq.) in portions and the solution stirred for an additional 2 hours atroom temperature. The reaction was quenched with 2 ml of acetone, 10 mlof CHCl₃ and 10 ml of 0.1N aqueous NaHCO₃ were added, the layers wereseparated, the organic layer was dried and evaporated to dryness to give704 mg (about 84%) of compound 39 which was used without additionalpurification. Compound 39 (838 mg or 1 mmol) was dissolved in a mixtureof CHCl₃ (5 ml) and diisophrophylethylamine (0.68 ml or 4 eq.). To thissolution was added 2-cyanoethyl-N-,N-diisophropylchlora-phosphoramidite(225 μl or 1 eq.) and the solution was stirred at room temperature for 1hour. Ethyl acetate (5 ml) was added and the solution was washed with anNaCl solution (3×1 ml) and H₂O (2 ml). Dried solvents were removed invacuo and purified on a silica gel column using step gradient oftriethylamine in CH₂Cl₂ with a yield of 789 mg (about 74%).

Example 8 Chemical Synthesis of Oligonucleotides usingPCB-Phosphoramidites

Automated Synthesis and Purification of Oligonucleotides Using 5′PCB-phosphoramidite: A 0.1M solution of 5′-PCB-phosphoramidite inanhydrous acetonitrile was prepared. The bottle with the solution wasplaced in the additional phosphoramidite port of the Applied Biosystem392 DNA/RNA synthesizer. An oligodeoxynucleotide sequence was programmedand synthesized using 40 nmol CE column and standard synthesis protocol.The only modification necessary was extended detritylation (180 s)necessary for removal of trityl group from N¹ position of biotin. Aftersynthesis, 5′-PCB-oligodeoxynucleotide was cleaved from solid supportand deprotected by treatment with concentrated ammonia for 16 hours at50° C. The crude oligonucleotide was freeze-dried and dissolved in 1 mlof phosphate buffer, pH 7.4. To this solution was added a suspension ofmonomeric avidin agarose beads (Sigma Chemical; St. Louis, Mo. ) (40nmoles), and the mixture was incubated at room temperature for 1 hour.The suspension was filtered and washed with 3×1 ml phosphate buffer,resuspended in 3 ml of phosphate buffer and illuminated as describedwith gentle stirring for 10 minutes. The mixture was filtered, filtratefreeze-dried and redissolved (yield=3.8 OD₂₆₀).

Example 9 Enzymatic Synthesis of DNA and RNA using PCB-Nucleotides

Several enzymatic and chemical methods are available for biotinylationof nucleic acid probes. Enzymatic methods for incorporation ofPCB-nucleotides into DNA include nick translation and replacementsynthesis using T4 DNA polymerase. Terminal labeling of DNA can also beperformed using terminal deoxynucleotidyl transferase. For PCB-labelingof RNA several RNA polymerase enzymes can be used. Nick translation wasperformed in the presence of PCB-dCTP based on the methods developed forbiotinylation (P. R. Langer et al., Proc. Natl. Acad. Sci. USA 78:6633,1981). Enzymatic tailing was used for double- and single-stranded DNAmolecules. PCB nucleotides were added onto the 3′-end of the DNA.Biotinylated probes with internal biotin moieties form less stablehybrids than probes with external biotins and that the biotinylatedprobes synthesized in this manner have greater sensitivity than probesthat are singly biotinylated at 5′-end (E. P. Diamandis et al., Clin.Chem. 37:625, 1991).

Preparation of PCB-labeled RNA: PCB-labeling of RNA was achieved in astandard phage T7 RNA polymerase transcription system using the PCB-UTP.To prepare single-stranded, biotinylated RNA as a probe, the appropriateDNA sequence was cloned into an appropriate vector which contains the T7promoter upstream from the polylinker region. After linearization of theDNA clone downstream from the cloned insert, the RNA transcript ofdefined length was produced by the T7 RNA polymerase using ATP, CTP, GTPand PCB-UTP as substrates.

Example 10 Isolation of Hematopoietic Cells for Autologous Bone MarrowTransplantation.

Bone marrow is collected from the posterior iliac crest of normalhealthy and leukemic patients into heparin. Low-density mononuclearcells are separated by sedimentation on Ficoll-Hypaque (Sigma Chemical;St. Louis, Mo.). CD34⁺ cells are isolated using PCB-labeled anti-CD34monoclonal antibodies (My10). Mononuclear marrow cells are placed atconcentrations of 10⁶/ml in Iscove's Modified Dulbeco's Medium (IMDM;Irvine Scientific; Santa Anna, Calif.) with 20% FCS. Cells are culturedovernight under tissue culture conditions to remove adherent cells.Nonadherent cells are collected, washed twice in cold phosphate bufferedsaline (PBS), and diluted in PBS to 10⁷/ml. PCB-labeled anti-CD34antibodies are added to the cell suspension at 5 μ/ml and incubated at4° C. for one hour with gentle intermittent mixing. After incubation,cells are washed twice in 5%-FCS/PBS and resuspended in the same volume.Streptavidin coated magnetic beads (Dynabeads; Oslo, Norway) are addedto the suspension which is incubated at 4° C. for one hour with mixing.Beads and their associated cells are subjected to a magnet and separatedfrom the suspension and placed in 5%-FCS/PBS. Photocleavage is carriedout by irradiating the beads for 4 minutes with a long-wavelength,UV-lamp (Black Ray XX-15 UV lamp; UVP Inc; San Gabriel, Calif.) at adistance of 15 cm (emission peak 365 nm, lamp intensity=1.1 mW/cm² at adistance of 31 cm). Released beads are isolated by magnetic capture. Thecell suspension is assayed for CD34⁺ cells by staining withFTC-conjugated My10 antibody followed with FACS analysis and determinedto be greater than 95% CD34 cells.

Example 11 Determination of the In Vivo Half-life of a PharmaceuticalComposition

Cell-free translation reactions are performed by mixing 10 μl ofPCB-coumarin amino acid-tRNA^(leu), prepared by chemicalmisaminoacylation as described above and suspended in TE at 1.7 mg/ml),50 μl of S-23 extract, 10 μl water and 10 μl of a solution of 50 mMmagnesium acetate, 50 mM Tris-acetate, pH 7.6, 200 mM Hepes-KOH buffer,pH 7.5; 1 M potassium acetate, 5 mM DTT, 3.75 mM GTP, 25 mM ATP, 100 mMcreatine phosphate and 600 μg/ml creatine kinase. This mixture is kepton ice until the addition of 20 μl of 500 μg/ml human IL-2 mRNA(containing 26 leucine codons) transcribed and isolated from recombinantIL-2 cDNA. The mixture is incubated at 25° C. for one hour and placed onice. One hundred μl of streptavidin coated magnetic Dynabeads (2 mg/ml)are added to the mixture which is placed at room temperature for 30minutes. After incubation, the mixture is centrifuged for 5 minutes in amicrofuge at 3,000×g or, a magnetic field is applied to the solutionusing a MPC. Supernatant is removed and the procedure repeated threetimes with TE. The final washed pellet is resuspended in 50 μl of 50 mMTris-HCl, pH 75 and transferred to a quartz cuvette. UV light from aBlack-Ray long wave UV lamp is applied to the suspension forapproximately one second. A magnetic field is applied to the solutionwith a MPC for 1.0 minute and the supernatant removed with a pipette.The supernatant is sterile filtered and mixed with equal volumes ofsterile buffer containing 50% glycerol, 1.8% NaCl and 25 mM sodiumbicarbonate. Protein concentration is determined by measuring theO.D.₂₆₀.

0.25 ml of the resulting composition is injected iv. into the tail veinof 2 Balb/c mice at concentrations of 1 mg/ml. Two control mice are alsoinjected with a comparable volume of buffer. At various time points (0,5 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours and 6 hours), 100μl serum samples are obtained from foot pads and added to 400 μl of 0.9%saline. Serum sample are added to a solution of dynabeads (2 mg/ml)coated with anti-coumarin antibody and incubated at room temperature for30 minutes. A magnetic field is applied to the solution with a MPC for 1minute and the supernatant removed with a pipette. Fluorescence at 470nm is measured and the samples treated with monoclonal antibody specificfor rat IL-2 protein. IL-2 protein content is quantitated for eachsample and equated with the amount of fluorescence detected. From theresults obtained, in vivo IL-2 half-life is accurately determined.

Example 12 Polymerase Chain Reactions with PCB

The steps involved in the PCR amplification DNA sequences using PCB areshown in FIG. 12. The experimental method described below is based on acombination of protocols described (Y. Lo et al., Nucl. Acids Res.16:719, 1988; R. K. Saiki et al., Sci. 239:487, 1988). PCB-dCTPsynthesized was added (50 μM) to the mixture of dATP, dGTP, dTTP (all200 μM) and dCTP (150 μM). The reaction mixture consisted of target DNAsource (total genomic DNA isolated), flanking primers and thethermostable polymerase (Taq polymerase). The reaction mixture wassubjected to 25-30 cycles of amplification. Samples were heated from70-95° C. for a 1 minute period to denature DNA and cooled to 40° C. for2 minutes to anneal the primers. Samples were again heated to 70° C. for1 minute to activate the polymerase and incubated at this temperaturefor 0.5 minutes to extend the annealed primers. After the last cycle,samples were incubated for an additional 5 to 10 minutes at 37° C. toensure that the final extension was complete. Magnetic capture of thenucleic acids was performed using streptavidin coated magnetic beads.The captured material was washed with appropriate buffers andresuspended at the desired concentration. Samples were illuminated for10 minutes to release the PCR product in an unmodified form.

Nucleic acids in either immobilized form or in solution form weredetected or separated (purified) using PCB-labeled nucleic acid probes.Hybridization was carried out to obtain DNA:DNA, DNA:RNA and RNA:RNAhybrids. These experiments involve first the hybridization withPCB-labeled probes followed by capturing the hybrids using streptavidincoated immobilized supports. These hybrids were washed free of initialundesired components and were released from the immobilized supportusing irradiation (G. Gebeyehu et al., Nucl. Acids Res. 15:4513, 1987;T. Ito et al., Nucl. Acids Res. 20:3524, 1992). Hybridization of ssDNAmolecules with PCB-probe involved incubation of these components in ahybridization buffer at 42° C. for 30 minutes. Hybridization conditionswere optimized for each probe and experimental system. PCB-labeledprobes had lower melting temperatures than radiolabeled probes andrequire slightly modified hybridization conditions. These hybrids wereselectively removed from the reaction components using immobilizedstreptavidin (Dynabeads M280 streptavidin). Photochemical release ofcomplexes resulted in the isolation of pure hybrid.

Example 13 Synthesis of PCB—Liposomes

Incorporation of PCB-Lipids into liposomes: PCB-lipids were mixed withconventional lipids in chloroform:methanol at a ratio of 2:1. The lipidmixture was evaporated to dryness under nitrogen and the dried lipidssuspended in DMSO as solvent to a final concentration of 1 mg/ml.Liposomes were sonication under nitrogen in an ice-cold chamber for 10minutes. The resulting suspension was centrifuged for 20 minutes at10,000 rpm and the supernatant containing PCB-liposomes was ready foruse. Satisfactory results were obtained with as little as 5% (molequivalent) PCB-lipids. The structural chemical formulas forPCB-phosphatidylathanolamine and PCB-phosphatidylserine are shown inFIG. 16.

Example 14 PCB for In Situ Hybridization

The general methodology for in situ hybridization reactions can bedivided into sample preparation, selection of indicator molecule andprobe, hybridization, washing, and autoradiography and detection.

Sample preparation: Frozen tissue sections of 5 to 6 μm are mounted ongelatin coated microscope slides and air dried for 30 min prior tofixation. This is followed by fixation of DNA or RNA using eitherglutaraldehyde or paraformaldehyde. During these fixing steps optionaldenaturing steps (e.g. 100° C. for 5 minutes) followed by quickimmersion in ice cold buffer (necessary if dsDNA or dsRNA is the targetof the reaction) can be introduced. In case of cells and cell-cultures,these cells (1×10⁶ cells) are deposited on gelatin coated slides bycytocentrifugation or smearing. The cells are air dried and fixed forDNA or RNA.

Selection of indicator molecule and probe: Although isotopic detectionoffers several advantages over the use of non-isotopic methods, thelatter can be used effectively. Labeled probes can be generated by avariety of techniques ranging from synthetic oligonucleotides to excisedplasmid inserts.

Hybridization: ISH follows the same general principles as a solution andfilter hybridization. Standard reaction temperatures are approximatelyT_(m)−25° C. (T_(m) is the temperature at which 50% of hybridsdissociate). The reaction temperature is reduced to a level compatiblewith the preservation of histological detail by the addition of 50%formamide to the hybridization mixture. Thus, for typical DNA-DNAhybridization reactions, the temperature is 37° C., for RNA-DNA 44° C.,and for RNA-RNA 50°. The surface of the microscope slide supporting thesample is gently blown by a stream of air, and the final hybridizationmix is pipetted over the surface. The film is then incubated flat in abath of paraffin oil for the required time and temperature.

Washing: The paraffin oil is drained and excess of oil is removed bywashing twice with chloroform, and the slides are air dried. A highstringency wash is given to reduce background.

Autoradiography and detection: Detection is carried out either usingX-ray film or emulsion coated cover-slips in cases of radioactiveisotopically labeled probes, and other methods in the case of enzymaticdetection methods.

Example 15 Isolation of Different Populations Cells with Agents whichPhotocleaved at Distinct Wavelengths

Two distinct conjugates are created, each with a differentantigen-specific antibody coupled to a different bioreactive agent.Conjugate A comprises compound 30 (FIG. 21), a PCB bioreactive agent,coupled to an antibody specific for the cell surface marker CD34 (a stemcell marker), and will photocleave with radiation at 300 nm. Conjugate Bcomprises compound 25 (FIG. 19), a PCB bioreactive agent, coupled to anantibody specific for the cell surface marker CD3 (a T cell marker), andwill photocleave with radiation at 400 nm.

Conjugates A and B are incubated, in duplicate, with samples ofperipheral blood obtained from healthy human volunteers. Incubations areperformed at room temperature (22° C.) with gentle rocking to providemaximal antibody-antigen contact. After a 30 minute incubation, cellsare placed in 100 mm tissue culture dishes coated with streptavidin andincubated for an additional 30 minutes. Upon streptavidin-biotinbinding, plates are gently washed in PBS to remove any cells which donot adhere.

After washing, one set of plates is treated with electromagneticradiation at 300 nm and the released cells collected. This set is thentreated with electromagnetic radiation at 400 nm and the cells releasedat this frequency collected. A second set of plates is treated withradiation at 400 nm, released cells are collected, the plates are againtreated at 300 nm and the released cells again collected. By determiningthe number of cells collected after each treatment and from each set ofplates, the number of cells in a sample of peripheral blood which carrythe cell surface marker for CD34, CD3, and both CD34 and CD3 isdetermined.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered exemplary only, with the true scope andspirit of the invention being indicated by the following claims.

1. A method, comprising: a) providing a tRNA molecule, nucleic acidderived from a biological sample, and a marker; b) aminoacylating saidtRNA molecule with said marker to create a misaminoacylated tRNA; c)introducing said misaminoacylated tRNA and said nucleic acid derivedfrom a biological sample into a translation system under conditions suchthat said marker is incorporated into a nascent protein; and d)screening for disease, said screening comprising detecting said marker.2. The method of claim 1, wherein the nascent protein is selected fromrecombinant gene products, gene fusion products, enzymes, cytokines,carbohydrate and lipid binding proteins, nucleic acid binding proteins,hormones, immunogenic proteins, human proteins, viral proteins,bacterial proteins, parasitic proteins and fragments and combinationsthereof.
 3. The method of claim 1, wherein the translation systemcomprises a cellular or cell free translation system.
 4. The method ofclaim 3, wherein the cellular translation system is selected from thegroup consisting of tissue culture cells, primary cells, cells in vivo,isolated immortalized cells, human cells and combinations thereof. 5.The method of claim 3, wherein the cell-free translation system isselected from the group consisting of Escherichia coli lysates, wheatgerm extracts, insect cell lysates, rabbit reticulocyte lysates, frogoocyte lysates, dog pancreatic lysates, human cell lysates, mixtures ofpurified or semi-purified translation factors and combinations thereof.6. The method of claim 1, wherein the tRNA molecule is aminoacylated bychemical or enzymatic misaminoacylation.
 7. The method of claim 1,wherein said disease is selected from the group consisting ofinfections, neoplasias and genetic disorders.
 8. The method of claim 1,wherein said biological sample is selected from the group consisting ofblood, serum, tissue, urine, stool, nasal cells and spinal fluid.
 9. Themethod of claim 1, wherein prior to step (c) said nucleic acid isamplied in a polymerase chain reaction.
 10. The method of claim 1,wherein said marker comprises a detectable label.
 11. The method ofclaim 1, wherein said marker comprises a coupling agent.
 12. The methodof claim 11, wherein said coupling agent is selected from the groupconsisting of biotin and derivatives thereof.
 13. The method of claim11, wherein said coupling agent comprises an antigenic site.
 14. Themethod of claim 11, wherein said coupling agent binds to surfaces, saidsurfaces selected from the group consisting of resin surfaces, beadsurfaces, ceramic surfaces, glass surfaces, particle surfaces, andpolymer surfaces.
 15. The method of claim 1, wherein said marker is aphotocleavable marker.
 16. The method of claim 15, wherein saidphotocleavable marker is photocleavable biotin.
 17. The method of claim16, wherein said photocleavable marker comprises an amino acid coupledvia a photocleavable linkage to a detectable label.
 18. The method ofclaim 15, wherein said photocleavable marker comprises an amino acidcoupled via a photocleavable linkage to a coupling agent.
 19. The methodof claim 1, wherein two or more different misaminoacylated tRNAs areintroduced to the translation system at step (c).
 20. The method ofclaim 1, wherein said detecting of step (d) comprises using anelectromagnetic field.
 21. A method of screening for a genetic disorder,comprising: a) providing a tRNA molecule, nucleic acid derived from abiological sample, and a detectable label; b) aminoacylating said tRNAmolecule with said detectable label to create a misaminoacylated tRNA;c) introducing said misaminoacylated tRNA and said nucleic acid derivedfrom a biological sample into a translation system under conditions suchthat said detectable label is incorporated into a nascent protein; andd) screening for a genetic disorder, said screening comprising detectingsaid detectable label.
 22. The method of claim 21, wherein the nascentprotein is selected from recombinant gene products, gene fusionproducts, enzymes, cytokines, carbohydrate and lipid binding proteins,nucleic acid binding proteins, hormones, immunogenic proteins, humanproteins, viral proteins, bacterial proteins, parasitic proteins andfragments and combinations thereof.
 23. The method of claim 21, whereinthe translation system comprises a cellular or cell-free translationsystem.
 24. The method of claim 23, wherein the cellular translationsystem is selected from the group consisting of tissue culture cells,primary cells, cells in vivo, isolated immortalized cells, human cellsand combinations thereof.
 25. The method of claim 23, wherein thecell-free translation system is selected from the group consisting ofEscherichia coli lysates, wheat germ extracts, insect cell lysates,rabbit reticulocyte lysates, frog oocyte lysates, dog pancreaticlysates, human cell lysates, mixtures of purified or semi-purifiedtranslation factors and combinations thereof.
 26. The method of claim21, wherein the tRNA molecule is aminoacylated by chemical or enzymaticmisaminoacylation.
 27. The method of claim 21, wherein said geneticdisorder is familial adenomatous polyposis.
 28. The method of claim 21,wherein said biological sample is selected from the group consisting ofblood, serum, tissue, urine, stool, nasal cells and spinal fluid. 29.The method of claim 21, wherein prior to step (c) said nucleic acid isamplied in a polymerase chain reaction.
 30. The method of claim 21,wherein said detectable label comprises a fluorescent label.
 31. Themethod of claim 21, wherein said detectable label comprises a couplingagent.
 32. The method of claim 31, wherein said coupling agent isselected from the group consisting of biotin and derivatives thereof.33. The method of claim 31, wherein said coupling agent comprises anantigenic site.
 34. The method of claim 31, wherein said coupling agentbinds to surfaces, said surfaces selected from the group consisting ofresin surfaces, bead surfaces, ceramic surfaces, glass surfaces,particle surfaces, and polymer surfaces.
 35. The method of claim 31,wherein two or more different misaminoacylated tRNAs are introduced tothe translation system at step (c).
 36. The method of claim 31, whereinsaid detecting of step (d) comprises using an electromagnetic field.